U.S. patent application number 13/030840 was filed with the patent office on 2011-06-16 for methods for treating and preventing fibrosis.
Invention is credited to Mary Collins, Michael J. Grusby, Thomas A. Wynn, Deborah A. YOUNG.
Application Number | 20110142833 13/030840 |
Document ID | / |
Family ID | 36741387 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110142833 |
Kind Code |
A1 |
YOUNG; Deborah A. ; et
al. |
June 16, 2011 |
METHODS FOR TREATING AND PREVENTING FIBROSIS
Abstract
The present invention provides methods of screening for
compositions useful for treating, ameliorating, or preventing
fibrosis and/or fibrosis-associated conditions by measuring changes
in the level(s) of IL-21 and/or IL-21 receptor (IL-21R) (e.g., the
level of expression of IL-21 and/or IL-21R protein and/or mRNA, the
level of activity of IL-21 and/or IL-21R, the level of interaction
of IL-21 with IL-21R). The invention further provides antagonists
of IL-21 or IL-21R for the treatment of fibrosis and/or
fibrosis-associated conditions. Further provided herein are methods
of diagnosing, prognosing, and monitoring the progress (e.g., the
course of treatment) of fibrosis and/or fibrosis-associated
conditions by measuring the level of IL-21 and/or IL-21R (i.e., the
level of activity of IL-21 and/or IL-21R, the level of expression
of IL-21 and/or IL-21R (e.g., the level of IL-21 and/or IL-21R gene
products), and/or the level of interaction of IL-21 with
IL-21R).
Inventors: |
YOUNG; Deborah A.; (Melrose,
MA) ; Wynn; Thomas A.; (Silver Spring, MD) ;
Collins; Mary; (Natick, MA) ; Grusby; Michael J.;
(Newton, MA) |
Family ID: |
36741387 |
Appl. No.: |
13/030840 |
Filed: |
February 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11402885 |
Apr 13, 2006 |
7910105 |
|
|
13030840 |
|
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|
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60671374 |
Apr 14, 2005 |
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Current U.S.
Class: |
424/133.1 ;
424/141.1; 424/649; 435/29; 514/18.6; 514/20.5; 514/21.2 |
Current CPC
Class: |
A61K 38/20 20130101;
Y02A 50/30 20180101; A61P 25/00 20180101; A61P 13/12 20180101; A61P
7/00 20180101; A61K 45/06 20130101; A61K 33/24 20130101; C07K
16/2866 20130101; A61P 29/00 20180101; G01N 33/6869 20130101; A61K
2039/505 20130101; A61P 9/10 20180101; G01N 2800/00 20130101; C07K
14/7155 20130101; Y02A 50/423 20180101; A61P 19/04 20180101; A61K
38/13 20130101; A61P 37/00 20180101; C07K 2319/30 20130101; C07K
16/244 20130101; A61P 27/02 20180101; A61P 21/00 20180101; A61K
38/1793 20130101; A61P 1/00 20180101; A61P 17/02 20180101; A61P
1/18 20180101; A61P 17/00 20180101; A61P 15/00 20180101; A61P 17/18
20180101; A61P 11/00 20180101; A61P 1/16 20180101; A61P 9/00
20180101; A61P 35/00 20180101; A61K 38/20 20130101; A61K 2300/00
20130101; A61K 38/13 20130101; A61K 2300/00 20130101; A61K 33/24
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
514/21.2; 514/18.6; 424/141.1; 514/20.5; 424/649; 435/29 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 39/395 20060101 A61K039/395; A61P 11/00 20060101
A61P011/00; A61P 1/16 20060101 A61P001/16; A61P 17/00 20060101
A61P017/00; A61P 29/00 20060101 A61P029/00; C12Q 1/02 20060101
C12Q001/02 |
Claims
1. A method for treating, ameliorating, or preventing fibrosis or a
fibrosis-associated disorder in a subject comprising administering
to the subject a therapeutically effective amount of an agent that
reduces the level of IL-21 and/or IL-21R in the subject.
2. The method of claim 1, wherein the agent is a soluble fragment
of an IL-21R.
3. The method of claim 2, wherein the soluble fragment of the
IL-21R comprises an amino acid sequence that is at least 90%
identical to an amino acid sequence selected from the group
consisting of amino acids 1-538 of SEQ ID NO:2, amino acids 20-538
of SEQ ID NO:2, amino acids 1-235 of SEQ ID NO:2, amino acids
20-235 of SEQ ID NO:2, amino acids 1-236 of SEQ ID NO:2, amino
acids 20-236 of SEQ ID NO:2, amino acids 1-529 of SEQ ID NO:5,
amino acids 20-529 of SEQ ID NO:5, amino acids 1-236 of SEQ ID
NO:5, and amino acid 20-236 of SEQ ID NO:5.
4. The method of claim 3, wherein the soluble fragment of the
IL-21R binds to an IL-21 polypeptide.
5. The method of claim 2, wherein the soluble fragment of the
IL-21R comprises an amino acid sequence that is substantially
identical to the amino acid sequence set forth in SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27.
6. The method of claim 5, wherein the amino acid sequence of the
soluble fragment of the IL-21R comprises an amino acid sequence
that is substantially identical to the amino acid sequence set
forth in SEQ ID NO:11 or SEQ ID NO:13.
7. The method of claim 2, wherein the soluble fragment of the
IL-21R is encoded by a nucleotide sequence that is substantially
identical to the nucleic acid sequence set forth in SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.
8. The method of claim 7, wherein the soluble fragment of the
IL-21R is encoded by a nucleotide sequence that is substantially
identical to the nucleic acid sequence set forth in SEQ ID NO:12 or
SEQ ID NO:16.
9. The method of claim 2, wherein the agent is a soluble fragment
of an IL-21R, and wherein the soluble fragment of the IL-21R
comprises an extracellular domain of IL-21R and an immunoglobulin
Fc fragment.
10. The method of claim 9, wherein the amino acid sequence of the
extracellular domain of the IL-21R comprises an amino acid sequence
that is at least 90% identical to amino acids 1-235 of SEQ ID NO:2
or amino acids 20-235 of SEQ ID NO:2.
11. The method of claim 9, wherein the immunoglobulin Fc fragment
has an altered function.
12. The method of claim 11, wherein the immunoglobulin Fc fragment
has the amino acid sequence of amino acids 244-467 of SEQ ID
NO:17.
13. The method of claim 1, wherein the fibrosis or
fibrosis-associated disorder affects the liver, epidermis,
endodermis, muscle, tendon, cartilage, heart, pancreas, lung,
uterus, nervous system, testis, ovary, adrenal gland, artery, vein,
colon, small intestine, biliary tract, or stomach.
14. The method of claim 13, wherein the fibrosis or
fibrosis-associated disorder affects the liver, epidermis,
endodermis, or lung.
15. The method of claim 14, wherein the fibrosis or
fibrosis-associated disorder is interstitial lung fibrosis.
16. The method of claim 13, wherein the fibrosis or
fibrosis-associated disorder is the result of an infection with
schistosoma.
17. The method of claim 1, wherein the fibrosis or
fibrosis-associated disorder is the result of wound healing.
18. The method of claim 17, wherein the wound healing results from
a surgical incision.
19. The method of claim 1, further comprising administering to the
subject at least one additional therapeutic agent.
20. The method of claim 19, wherein the at least one additional
therapeutic agent is selected from the group consisting of cytokine
inhibitors, growth factor inhibitors, immunosuppressants,
anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors,
cytotoxic agents, and cytostatic agents.
21. The method of claim 19, wherein the at least one additional
therapeutic agent is selected from the group consisting of TNF
antagonists, anti-TNF agents, IL-12 antagonists, IL-15 antagonists,
IL-17 antagonists, IL-18 antagonists, IL-22 antagonists, T
cell-depleting agents, B cell-depleting agents, cyclosporin, FK506,
CCI-779, etanercept, infliximab, rituximab, adalimumab,
prednisolone, azathioprine, gold, sulphasalazine,
hydroxychloroquine, minocycline, anakinra, abatacept, methotrexate,
leflunomide, rapamycin, rapamycin analogs, Cox-2 inhibitors, cPLA2
inhibitors, NSAIDs, p38 inhibitors, antagonists of B7.1, B7.2,
ICOSL, ICOS and/or CD28, and agonists of CTLA4.
22. The method of claim 1, wherein the subject is a human.
23. A method for identifying a compound for treating, ameliorating
or preventing fibrosis or a fibrosis-associated disorder in a
subject, comprising: (a) measuring the level of IL-21 and/or IL-21R
in a cell or sample of interest; (b) contacting the cell or sample
of interest with a compound; and (c) measuring the level of IL-21
and/or IL-21R in the cell or sample of interest following contact
with the compound, wherein a lower level of IL-21 and/or IL-21R in
the contacted cell or sample of interest, in comparison to the
level of IL-21 and/or IL-21R in a noncontacted cell or sample of
interest, identifies the compound as a compound useful for
treating, ameliorating, or preventing fibrosis or a
fibrosis-associated condition in a subject.
24. A method for identifying a compound for treating, ameliorating
or preventing fibrosis or a fibrosis-associated disorder in a
subject, comprising: (a) measuring the level of IL-21 and/or IL-21R
in a cell or sample of interest; (b) contacting the cell or sample
of interest with a compound; (c) measuring the level of IL-21
and/or IL-21R in the cell or sample of interest following contact
with the compound; and (d) comparing the level of IL-21 and/or
IL-21R in the contacted cell or sample of interest with a reference
level of IL-21 and/or IL-21R, wherein a lower level of IL-21 and/or
IL-21R in the contacted cell or sample of interest, in comparison
to the reference level of IL-21 and/or IL-21R, identifies the
compound as a compound useful for treating, ameliorating, or
preventing fibrosis or a fibrosis-associated condition in a
subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 11/402,885, filed Apr. 13, 2006,
entitled "METHODS FOR TREATING AND PREVENTING FIBROSIS," which
claims priority to U.S. Provisional Application No. 60/671,374,
filed Apr. 14, 2005. The contents of both applications are
incorporated by reference herein in their entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] The Sequence Listing filed on Feb. 18, 2011, created on Feb.
18, 2011, named 019970438001ST25.txt, having a size in bytes of 116
kb, is hereby incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods for treating and
preventing fibrosis and fibrosis-associated conditions.
[0005] 2. Related Background Art
[0006] Injury to any organ typically leads to a physiological
response involving platelet-induced hemostasis, followed by an
influx of inflammatory cells and activated fibroblasts. Cytokines
produced by these cell types drive the formation of new
extracellular matrix and blood vessels, which collectively form
granulation tissue. The formation of fibrous tissue is part of the
normal beneficial process of healing following injury; fibrosis,
however, is a condition characterized by an abnormal accumulation
of a collagen matrix following injury or inflammation that alters
the structure and function of various tissues. Progressive fibrosis
in the kidney, liver, lung, heart, bone, bone marrow, and skin is a
major cause of, or contributor to, death.
[0007] Many of the diseases associated with the proliferation of
fibrous tissue are chronic and often debilitating and include, for
example, skin diseases such as scleroderma. Some, including
pulmonary fibrosis, may be fatal, due in part to the fact that the
current treatments have significant side effects and are generally
not effective in slowing or halting the progression of fibrosis.
There is, accordingly, a continuing need for new anti-fibrotic
agents.
[0008] The IL-21 receptor (IL-21R) is a newly discovered member of
the class I cytokine receptor family (Parrish-Novak et al. (2000)
Nature 408:57-63; Ozaki et al. (2000) Proc. Natl. Acad. Sci. U.S.A.
97:11439-44). The IL-21 receptor shows significant sequence and
structural homology with the IL-4 receptor alpha (IL-4R.alpha.)
chain and is adjacent to the IL-4R.alpha. in the human and mouse
genomes, while its ligand, IL-21, shares significant homology with
the cytokines IL-2, IL-4, and IL-15 (Sivakumar et al. (2004)
Immunology 112:177-82; Habib et al. (2003) J. Allergy Clin.
Immunol. 112:1033-45). IL-21 and IL-21R are thus newly described
members of the gamma chain (.gamma.c)-dependent cytokine network
because of their homology with cytokines and receptors that require
the .gamma.c for functional signaling (Vosshenrich and Di Santo
(2001) Curr. Biol. 11:R175-77). Because all members of the .gamma.c
network exhibit important and unique roles in host immunity, there
has been growing interest in dissecting the novel functions of the
IL-21R during antigen-triggered immune responses in vivo.
[0009] Initial studies examining the function of IL-21 showed that
NK cell expansion is antagonized, whereas antigen-specific T cell
immunity is promoted by IL-21, including anti-tumor immunity (Ma et
al. (2003) J. Immunol. 171:608-15; Kishida et al. (2003) Mol. Ther.
8:552-58; Di Carlo et al. (2004) J. Immunol. 172:1540-47), findings
that suggest that IL-21 serves as a bridge between innate and
adaptive immune responses (Collins et al. (2003) Immunol. Res.
28:131-40). IL-21 also regulates B cell and CD8.sup.+ T cell
function in vivo (Ozaki et al. (2002) Science 298:1630-34; Suto et
al. (2002) Blood 100:4565-73; Mehta et al. (2003) J. Immunol.
170:4111-18; Pene et al. (2004) J. Immunol. 172:5154-57; Jin et al.
(2004) J. Immunol. 173:657-65; Zeng et al. (2005) J. Exp. Med.
201:139-48). Additional studies suggest that IL-21 is a T.sub.H2
cytokine that can inhibit the differentiation of naive T.sub.H
cells into IFN-.gamma.-secreting T.sub.H1 cells (Wurster et al.
(2002) J. Exp. Med. 196:969-77). Indeed, exogenous treatment with
IL-21 potently inhibited IFN-.gamma. production without affecting
other T.sub.H1/T.sub.H2-associated cytokines, suggesting that the
repression of IFN-.gamma. by IL-21 is highly specific. Thus, by
virtue of its ability to suppress the development of T.sub.H1
cells, it was hypothesized that IL-21 might promote T.sub.H2
responses (Wurster et al., supra). Nevertheless, the involvement of
the IL-21R signaling pathway in T.sub.H2 response development was
not previously investigated in any T.sub.H2-dependent disorder.
[0010] In schistosomiasis, T.sub.H2 cytokines play an indispensable
role in the pathogenesis of the disease (Wynn (2004) Nat. Rev.
Immunol. 4:583-94; Pearce and MacDonald (2002) Nat. Rev. Immunol.
2:499-511). Indeed, IL-4/IL-13-, IL-4R.alpha.-, and Stat6-deficient
mice all show significantly impaired granuloma formation and liver
fibrosis following infection with S. mansoni (Chiaramonte et al.
(1999) J. Clin. Invest. 104:777-85; Kaplan et al. (1998) J.
Immunol. 160:1850-56; Jankovic et al. (1999) J. Immunol.
163:337-42; Fallon et al. (2000) J. Immunol. 164:2585-91). Given
the recent classification of IL-21 as a T.sub.H2 cytokine (Wurster
et al. (2002), supra; Mehta et al. (2005) Proc. Natl. Acad. Sci.
U.S.A. 102:2016-21), the striking similarities between the IL-4 and
IL-21 receptors (Sivakumar et al., supra; Habib et al., supra), and
the critical role of the related IL-4R.alpha./Stat6-signaling
pathway in this disease as well as in other T.sub.H2
cytokine-driven inflammatory disorders (Wynn (2003) Annu. Rev.
Immunol. 21:425-56), an important question evolving from these
studies was whether IL-21R signaling plays a significant role in
the initiation and/or maintenance of T.sub.H2 immunity.
SUMMARY OF THE INVENTION
[0011] The present invention provides methods of treating,
ameliorating or preventing fibrosis or a fibrosis-associated
disorder, as well as methods of screening for compounds and
compositions useful in those methods. The invention also provides
methods of diagnosing, prognosing, and monitoring the progress
(e.g., the course of treatment) of fibrosis and/or
fibrosis-associated conditions. These methods are related to
measuring and/or modulating the level of IL-21 and/or IL-21R (i.e.,
the level of activity of IL-21 and/or IL-21R, the level of
expression of IL-21 and/or IL-21R (e.g., the level of IL-21 and/or
IL-21R gene products), and/or the level of interaction of IL-21
with IL-21R). The invention further provides antagonists of IL-21
or IL-21R for the treatment of fibrosis and/or fibrosis-associated
conditions.
[0012] In one embodiment, the invention provides a method for
treating, ameliorating, or preventing fibrosis or a
fibrosis-associated disorder in a subject (e.g., a human)
comprising administering to the subject a therapeutically effective
amount of an agent that reduces the level of IL-21 and/or IL-21R in
the subject. In a further embodiment, the agent is an IL-21/IL-21R
antagonist selected from the group consisting of an anti-IL-21R
antibody, an anti-IL-21 antibody, an antigen-binding fragment of an
anti-IL-21R antibody, an antigen-binding fragment of an anti-IL-21
antibody, and a soluble fragment of an IL-21R. In another further
embodiment, the agent is a soluble fragment of an IL-21R, and the
soluble fragment of the IL-21R comprises an amino acid sequence
that is at least 90% identical to an amino acid sequence selected
from the group consisting of amino acids 1-538 of SEQ ID NO:2,
amino acids 20-538 of SEQ ID NO:2, amino acids 1-235 of SEQ ID
NO:2, amino acids 20-235 of SEQ ID NO:2, amino acids 1-236 of SEQ
ID NO:2, amino acids 20-236 of SEQ ID NO:2, amino acids 1-529 of
SEQ ID NO:5, amino acids 20-529 of SEQ ID NO:5, amino acids 1-236
of SEQ ID NO:5, and amino acid 20-236 of SEQ ID NO:5. In another
embodiment, the soluble fragment of the IL-21R binds to an IL-21
polypeptide.
[0013] In another embodiment, the agent is a soluble fragment of an
IL-21R, and the soluble fragment of the IL-21R comprises an amino
acid sequence that is substantially identical to the amino acid
sequence set forth in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,
or SEQ ID NO:27. In another embodiment, the amino acid sequence of
the soluble fragment of the IL-21R comprises an amino acid sequence
that is substantially identical to the amino acid sequence set
forth in SEQ ID NO:11 or SEQ ID NO:13. In another embodiment, the
agent is a soluble fragment of an IL-21R, and the soluble fragment
of the IL-21R is encoded by a nucleotide sequence that is
substantially identical to the nucleic acid sequence set forth in
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.
In another embodiment, the soluble fragment of the IL-21R is
encoded by a nucleotide sequence that is substantially identical to
the nucleic acid sequence set forth in SEQ ID NO:12 or SEQ ID
NO:16.
[0014] In another embodiment, the agent is a soluble fragment of an
IL-21R, and the soluble fragment of the IL-21R comprises an
extracellular domain of IL-21R and an immunoglobulin Fc fragment.
In a further embodiment, the amino acid sequence of the
extracellular domain of the IL-21R comprises an amino acid sequence
that is at least 90% identical to amino acids 1-235 of SEQ ID NO:2
or amino acids 20-235 of SEQ ID NO:2. In another embodiment, the
immunoglobulin Fc fragment has an altered function. In another
further embodiment, the immunoglobulin Fc fragment has the amino
acid sequence of amino acids 244-467 of SEQ ID NO:17.
[0015] In another embodiment, the fibrosis or fibrosis-associated
disorder affects the liver, epidermis, endodermis, muscle, tendon,
cartilage, heart, pancreas, lung, uterus, nervous system, testis,
ovary, adrenal gland, artery, vein, colon, small intestine, biliary
tract, or stomach. In a further embodiment, the fibrosis or
fibrosis-associated disorder is interstitial lung fibrosis. In
another embodiment, the fibrosis or fibrosis-associated disorder is
the result of an infection with schistosoma. In another embodiment,
the fibrosis or fibrosis-associated disorder is the result of wound
healing. In a further embodiment, the wound healing results from a
surgical incision.
[0016] In another embodiment, the invention further comprises
administering to the subject at least one additional therapeutic
agent. In another embodiment, the at least one additional
therapeutic agent is selected from the group consisting of cytokine
inhibitors, growth factor inhibitors, immunosuppressants,
anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors,
cytotoxic agents, and cytostatic agents. In a further embodiment,
the at least one additional therapeutic agent is selected from the
group consisting of TNF antagonists, anti-TNF agents, IL-12
antagonists, IL-15 antagonists, IL-17 antagonists, IL-18
antagonists, IL-22 antagonists, T cell-depleting agents, B
cell-depleting agents, cyclosporin, FK506, CCl-779, etanercept,
infliximab, rituximab, adalimumab, prednisolone, azathioprine,
gold, sulphasalazine, hydroxychloroquine, minocycline, anakinra,
abatacept, methotrexate, leflunomide, rapamycin, rapamycin analogs,
Cox-2 inhibitors, cPLA2 inhibitors, NSAIDs, p38 inhibitors,
antagonists of B7.1, B7.2, ICOSL, ICOS and/or CD28, and agonists of
CTLA4.
[0017] In another embodiment, the invention provides a method for
identifying a compound for treating, ameliorating or preventing
fibrosis or a fibrosis-associated disorder in a subject,
comprising: (a) measuring the level of IL-21 and/or IL-21R in a
cell or sample of interest; (b) contacting the cell or sample of
interest with a compound; and (c) measuring the level of IL-21
and/or IL-21R in the cell or sample of interest following contact
with the compound, wherein a lower level of IL-21 and/or IL-21R in
the contacted cell or sample of interest, in comparison to the
level of IL-21 and/or IL-21R in a noncontacted cell or sample of
interest, identifies the compound as a compound useful for
treating, ameliorating, or preventing fibrosis or a
fibrosis-associated condition in a subject.
[0018] In another embodiment, the invention provides a method for
identifying a compound for treating, ameliorating or preventing
fibrosis or a fibrosis-associated disorder in a subject,
comprising: (a) measuring the level of IL-21 and/or IL-21R in a
cell or sample of interest; (b) contacting the cell or sample of
interest with a compound; (c) measuring the level of IL-21 and/or
IL-21R in the cell or sample of interest following contact with the
compound; and (d) comparing the level of IL-21 and/or IL-21R in the
contacted cell or sample of interest with a reference level of
IL-21 and/or IL-21R, wherein a lower level of IL-21 and/or IL-21R
in the contacted cell or sample of interest, in comparison to the
reference level of IL-21 and/or IL-21R, identifies the compound as
a compound useful for treating, ameliorating, or preventing
fibrosis or a fibrosis-associated condition in a subject.
[0019] In another embodiment, the invention provides a method for
monitoring the progress of fibrosis or a fibrosis-associated
condition in a subject, comprising: (a) measuring the level of
IL-21 and/or IL-21R in a cell or sample of interest from the
subject at a first time point; and (b) measuring the level of IL-21
and/or IL-21R in a cell or sample of interest from the subject at a
second time point, wherein a lower level of IL-21 and/or IL-21R in
the cell or sample of interest from the subject at the second time
point, in comparison to the level of IL-21 and/or IL-21R in the
cell or sample of interest from the subject at the first time
point, provides an indication that the fibrosis or
fibrosis-associated condition has decreased in severity.
[0020] In another embodiment, the invention provides a method for
prognosing fibrosis or a fibrosis-associated condition in a
subject, comprising: (a) measuring the level of IL-21 and/or IL-21R
in a cell or sample of interest from the subject at a first time
point; and (b) measuring the level of IL-21 and/or IL-21R in a cell
or sample of interest from the subject at a second time point,
wherein a lower level of IL-21 and/or IL-21R in the cell or sample
of interest from the subject at the second time point, in
comparison to the level of IL-21 and/or IL-21R in the cell or
sample of interest from the subject at the first time point,
indicates a decreased likelihood that the subject will develop
fibrosis or the fibrosis-associated condition or a decreased
likelihood that the fibrosis or fibrosis-associate condition will
worsen in the subject.
[0021] In another embodiment, the invention provides a method for
prognosing fibrosis or a fibrosis-associated condition in a
subject, comprising: (a) measuring the level of IL-21 and/or IL-21R
in a cell or sample of interest from the subject; and (b) comparing
the level of IL-21 and/or IL-21R in the cell or sample of interest
from the subject to a reference level of IL-21 and/or IL-21R,
wherein a lower level of IL-21 and/or IL-21R in the cell or sample
of interest from the subject, in comparison to the reference level
of IL-21 and/or IL-21R, indicates a decreased likelihood that the
subject will develop fibrosis or the fibrosis-associated condition
or a decreased likelihood that the fibrosis or fibrosis-associate
condition will worsen in the subject.
[0022] In another embodiment, the invention provides a method for
diagnosing fibrosis or a fibrosis-associated condition in a
subject, comprising: (a) measuring the level of IL-21 and/or IL-21R
in a cell or sample of interest from the subject, and (b) comparing
the level of IL-21 and/or IL-21R in the cell or sample of interest
from the subject with a reference level of IL-21 and/or IL-21R,
wherein a higher level of IL-21 and/or IL-21R in the cell or sample
of interest from the subject, in comparison to the reference level
of IL-21 and/or IL-21R, indicates the presence of fibrosis or the
fibrosis-associated condition in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts the full-length cDNA sequence of murine
IL-21R/MU-1. The nucleotide sequence corresponds to nucleotides
1-2628 of SEQ ID NO:4. FIG. 1A depicts the cDNA sequence of murine
IL-21R/MU-1 from nucleotide 1 to 1450, and FIG. 1B depicts the cDNA
sequence of murine IL-21R/MU-1 from nucleotide 1451 to 2628.
[0024] FIG. 2 depicts the amino acid sequences of murine and human
IL-21R/MU-1. FIG. 2A depicts the amino acid sequence of murine
IL-21R/MU-1 (corresponding to the amino acids 1-529 of SEQ ID
NO:5). There is a leader sequence (predicted by SPScan with a score
of 10.1) at amino acids 1-19 (boldface type). A predicted
transmembrane domain (underlined) is found at amino acids 237-253
of SEQ ID NO:5. Predicted signaling motifs include the "Box 1"
motif at amino acids 265-274 and the "Box 2" motif at amino acids
311-324 (bold and underlined). Six tyrosines are located at amino
acid positions 281, 319, 361, 369, 397, and 510, of SEQ ID NO:5.
The WSXWS motif (SEQ ID NO:3) is located at amino acid residues
214-218 (in large, boldface type). Potential Stat docking sites
include amino acids 393-398 and amino acids 510-513 of SEQ ID NO:5.
FIG. 2B depicts the amino acid sequence of human IL-21R/MU-1
(corresponding to SEQ ID NO:2). The location of the predicted
signal sequence (about amino acids 1-19 of SEQ ID NO:2); WSXWS
motif (about amino acids 214-218 of SEQ ID NO:2); and transmembrane
domain (about amino acids 236-252 (or 236-253, or 236-254) of SEQ
ID NO:2 (underlined)) are indicated. Potential JAK binding sites,
Box 1 and 2 signaling motifs, and Stat docking sites are indicated
by labeled arrows.
[0025] FIG. 3 depicts the GAP comparison of human and murine
IL-21R/MU-1 cDNA sequences (corresponding to nucleic acids 1-1909,
1960-2050, and 2151-2665 of SEQ ID NO:1 and nucleic acids 151-2628
of SEQ ID NO:4, respectively). huMU-1=human IL-21R/MU-1,
murMU-1=murine IL-21R/MU-1. Gap Parameters Gap Weight=50; Average
Match=10.000; Length Weight=3; Average Mismatch=0.000; Percent
Identity=66.116. FIG. 3A is a comparison of nucleotides 1-721 of
human IL-21R/MU-1 of SEQ ID NO:1 to nucleotides 151-892 of mouse
IL-21R/MU-1 of SEQ ID NO:4; FIG. 3B is a comparison of nucleotides
722-1509 of human IL-21R/MU-1 of SEQ ID NO:1 to nucleotides
893-1674 of mouse IL-21R/MU-1 of SEQ ID NO:4; FIG. 3C is a
comparison of nucleotides 1510-2404 of human IL-21R/MU-1 of SEQ ID
NO:1, wherein nucleic acids 1910-1959 and 2051-2150 of SEQ ID NO:1
have been removed for the purposes of the alignment, to nucleotides
1675-2368 of mouse IL-21R/MU-1 of SEQ ID NO:4; and FIG. 3D is a
comparison of nucleotides 2405-2665 of human IL-21R/MU-1 of SEQ ID
NO:1 to nucleotides 2369-2628 of mouse IL-21R/MU-1 of SEQ ID
NO:4.
[0026] FIG. 4 depicts a GAP comparison of human IL-21R/MU-1 protein
(corresponding to amino acids of SEQ ID NO:2) and murine
IL-21R/MU-1 protein (corresponding to amino acids of SEQ ID NO:5).
The alignment was generated by BLOSUM62 amino acid substitution
matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. U.S.A.
89:10915-19). Gap parameters=Gap Weight: 8; Average Match=2.912;
Length Weight=2; Average Mismatch=-2.003; Percent
Identity=65.267.
[0027] FIG. 5 depicts a multiple sequence alignment of the amino
acids of human IL-21R/MU-1 (corresponding to SEQ ID NO:2), murine
IL-21R/MU-1 (corresponding to SEQ ID NO:5), and human IL-2 beta
chain (GENBANK.RTM. Accession No. M26062, corresponding to SEQ ID
NO:35). Leader and transmembrane domains are underlined. Conserved
cytokine receptor module motifs are indicated by boldface type.
Potential signaling regions are indicated by underlining and
boldface type.
[0028] FIG. 6 depicts signaling through IL-21R/MU-1. IL-21R/MU-1
phosphorylates Stat 5 in Clone E7 EPO IL-21R/MU-1 chimera
expressing cells stimulated with EPO. Treatment of controls or
chimeric BAF-3 cells with IL-3 resulted in phosphorylation of Stat
3, but not Stat 1 or 5.
[0029] FIG. 7 depicts an alignment of the nucleotide and amino acid
sequences of mature human IL-21R fused at the amino terminal to a
honeybee leader sequence and His.sub.6 and Flag tags. The
nucleotide and amino acid sequences of the fusion protein depicted
in FIGS. 7A-7B are set forth in SEQ ID NO:10 and SEQ ID NO:11,
respectively. The amino acid sequences of the mature human IL-21R
fragment and the honeybee leader/His tags fragment of the fusion
protein correspond to amino acids 20-235 of SEQ ID NO:2 and amino
acids 1-44 of SEQ ID NO:11, respectively.
[0030] FIG. 8 depicts an alignment of the nucleotide and amino acid
sequences of human IL-21R extracellular domain fused at the
C-terminus via a linker to human immunoglobulin G1 (IgG1) Fc
sequence. The nucleotide and amino acid sequences of the fusion
protein depicted in FIGS. 8A-8C are set forth in SEQ ID NO:12 and
SEQ ID NO:13, respectively. The amino acid sequences of the human
IL-21R extracellular domain, the linker, and the human
immunoglobulin G1 (IgG1) Fc sequence correspond to amino acids
1-235 of SEQ ID NO:2, amino acids 236-243 of SEQ ID NO:13, and
amino acids 244-467 of SEQ ID NO:13, respectively.
[0031] FIG. 9 depicts an alignment of the nucleotide and amino acid
sequences of human IL-21R extracellular domain fused at the
C-terminus via a linker to human immunoglobulin G1 (IgG1) Fc
sequence and His.sub.6 sequence tag. The nucleotide and amino acid
sequences of the fusion protein depicted in FIGS. 9A-9C are set
forth in SEQ ID NO:14 and SEQ ID NO:15, respectively. The amino
acid sequences of the human IL-21R extracellular domain, the
linker, the human immunoglobulin G1 (IgG1) Fc sequence, and the
His.sub.6 sequence tag correspond to amino acids 1-235 of SEQ ID
NO:2, amino acids 236-243 of SEQ ID NO:15, amino acids 244-467 of
SEQ ID NO:15, and amino acids 468-492 of SEQ ID NO:15,
respectively.
[0032] FIG. 10 depicts an alignment of the nucleotide and amino
acid sequences of human IL-21R extracellular domain fused at the
C-terminus via a linker to human immunoglobulin G1 (IgG1)
Fc-mutated sequence. The human Fc sequence has been mutated at
residues 254 and 257 from the wild-type sequence to reduce
Fc-receptor binding. The nucleotide and amino acid sequences of the
fusion protein depicted in FIGS. 10A-10C are set forth in SEQ ID
NO:16 and SEQ ID NO:17, respectively. The amino acid sequences of
the human IL-21R extracellular domain, the linker, and the human
immunoglobulin G1 (IgG1) Fc-mutated sequence correspond to amino
acids 1-235 of SEQ ID NO:2, amino acids 236-243 of SEQ ID NO:17,
and amino acids 244-467 of SEQ ID NO:17, respectively.
[0033] FIG. 11 depicts an alignment of the nucleotide and amino
acid sequences of human IL-21R extracellular domain fused at the
C-terminus to a rhodopsin sequence tag. The nucleotide and amino
acid sequences of the fusion protein depicted in FIGS. 11A and 11B
are set forth in SEQ ID NO:18 and SEQ ID NO:19, respectively. The
amino acid sequence of the human IL-21R extracellular domain
corresponds to amino acids 1-235 of SEQ ID NO:2.
[0034] FIG. 12 depicts an alignment of the nucleotide and amino
acid sequences of human IL-21R extracellular domain fused at the
C-terminus to an EK cleavage site and mutated IgG1 Fc region. The
nucleotide and amino acid sequences of the fusion protein depicted
in FIGS. 12A-12C are set forth in SEQ ID NO:20 and SEQ ID NO:21,
respectively. The amino acid sequences of the human IL-21R
extracellular domain, and the EK cleavage site/mutated IgG1 Fc
region correspond to amino acids 1-235 of SEQ ID NO:2 and amino
acids 236-470 of SEQ ID NO:21, respectively.
[0035] FIG. 13 depicts an alignment of the nucleotide and amino
acid sequences of murine IL-21R extracellular domain fused at the
C-terminus to mouse immunoglobulin G2a (IgG2a). The nucleotide and
amino acid sequences depicted in FIGS. 13A and 13B are set forth in
SEQ ID NO:22 and SEQ ID NO:23, respectively.
[0036] FIG. 14 depicts an alignment of the nucleotide and amino
acid sequences of murine IL-21R extracellular domain fused at the
C-terminus to Flag and His.sub.6 sequence tags. The nucleotide and
amino acid sequences depicted in FIGS. 14A and 14B are set forth in
SEQ ID NO:24 and SEQ ID NO:25, respectively.
[0037] FIG. 15 depicts an alignment of the nucleotide and amino
acid sequences of (honeybee leader) murine IL-21R extracellular
domain fused at the N-terminus to Flag and His.sub.6 sequence tags.
The nucleotide and amino acid sequences depicted in FIGS. 15A and
15B are set forth in SEQ ID NO:26 and SEQ ID NO:27,
respectively.
[0038] FIG. 16 shows IL-21 and IL-21R expression profiles during
highly polarized type-1 and type-2 immune responses. Groups of five
IL-10/IL-4 KO (TH1, .DELTA.) and IL-10/IL-12 KO (TH2, ) mice were
sensitized i.p. with S. mansoni eggs and challenged i.v. 14 days
later. Lung RNA specimens were prepared individually for real-time
RT-PCR analysis of IL-13 and IFN-.gamma. (FIG. 16A) and IL-21R and
IL-21 (FIG. 16B). The means.+-.SEM in gene expression were
expressed as fold-increases over unchallenged WT controls after
normalization to HPRT. Asterisks denote significant differences
between groups at the given time point, * p<0.05.
[0039] FIG. 17 shows that type-2 cytokine production is reduced in
the lungs of schistosome egg-challenged IL-21R.sup.-/- mice. Groups
of naive WT (open bars) and IL-21R.sup.-/- mice (filled bars) were
i.v. challenged with live S. mansoni eggs and sacrificed on days 4,
7, and 14 post-challenge. (FIG. 17A) RNA was prepared from lung
tissues and analyzed individually (N=5 per group/time point) by
real-time RT-PCR. Results are shown as box-and-whisker plots with
five-number summary bars showing the median, the quartiles, and the
smallest and greatest percentiles in the distribution; bars (from
bottom to top) indicate the 10th, 25th, 50th, 75th, and 90th
percentiles, respectively, of the tested samples. The asterisks
denote significant differences from wild-type values at the given
time point, * p<0.05, ** p<0.01, *** p<0.001. (FIG. 17B)
Spleens (Spl) and lung-associated lymph nodes (LN) were each pooled
(2 separate groups, 3-4 mice per group) and single cell suspensions
were assayed for IL-5, IL-10, IL-13, and IFN-.gamma. after a 72-h
incubation in the presence of Con A (CON, 1 .mu.g/ml) or soluble
egg antigen (SEA, 20 .mu.g/ml). Cytokines were below the level of
detection in unstimulated cultures. (FIG. 17C) Granuloma size
(volume, mm.sup.3.times.10.sup.-3) and the percentage of
eosinophils in granulomas were quantified microscopically. (FIG.
17D) Real-time PCR analysis of T.sub.H2-regulated inflammatory
genes in granulomatous lung tissue. All data are representative of
at least 2 separate experiments.
[0040] FIG. 18 shows that the type-2 response is impaired in N.
brasiliensis-infected IL-21R.sup.-/- mice. Lungs (FIG. 18A) and
lung-associated lymph nodes (LALN) (FIG. 18B) were removed on day 7
from individual N. brasiliensis-infected and untreated C57BL/6 or
IL-21R.sup.-/- mice (5/treatment group). RNA was isolated and cDNA
was generated as described in the legend to FIG. 17. mRNA was
analyzed individually for IL-13, IL-4, AMCase, Ym1, and FIZZ1 by
real-time quantitative PCR. Fold changes are based on comparisons
of infected mice to naive animals.
[0041] FIG. 19 shows that type-2 cytokine-driven inflammation is
reduced in IL-21R.sup.-/- mice. WT (open bars) and IL-21R.sup.-/-
mice (filled bars) were sensitized i.p. with eggs, challenged i.v.
two weeks later with live S. mansoni eggs and then sacrificed on
days 4 and 7 post-challenge. (FIG. 19A) RNA was prepared from lung
tissues and analyzed individually (N=5 per group/time point) by
real-time RT-PCR as described above in the legend to FIG. 17. (FIG.
19B) Spleens (Spl) and lung-associated lymph nodes (LN) were
assayed for IL-5, IL-10, IL-13, and IFN-.gamma. following antigen
(SEA) or mitogen stimulation (CON). (FIG. 19C) Granuloma size
(mm.sup.3.times.10.sup.-3) and the percentage of eosinophils in
granulomas were quantified microscopically in WT (mice per group:
N=10, day 4; N=15, day 7) and IL-21R.sup.-/- mice (N=11, day 4;
N=16, day 7). (FIG. 19D) Real-time PCR analysis of T.sub.H2
inflammatory genes in granulomatous lung tissue (N=5 per group/time
point). The asterisks denote significant differences from wild-type
values at the given time point, * p<0.05, ** p<0.01, ***
p<0.001. Data shown are the combined results of three separate
experiments.
[0042] FIG. 20 shows that chronic liver disease following
percutaneous S. mansoni infection is reduced in the absence of
IL-21R. WT (open bars) and IL-21R.sup.-/- mice (filled bars) were
infected with 25-30 S. mansoni cercariae. All animals were
sacrificed at week 9 (acute) or week 12 (chronic) post-infection.
(FIG. 20A) RNA was isolated from liver tissues and analyzed
individually (N=8-10 per group/time point) by real-time RT-PCR as
described above in the legend to FIG. 17. (FIG. 20B) Spleens (Spl)
and mesenteric lymph nodes (LN) were pooled in groups of 2-4 mice
and single cell suspensions were assayed for IL-5, IL-10, and
IFN-.gamma.. The data shown are the averages of three separate
pooled groups. (FIG. 20C) Granuloma size (mm.sup.3.times.10.sup.-3)
and the percentage of eosinophils in granulomas were evaluated
microscopically in WT mice (mice per group: N=30 for week 9, N=17
for week 12) and IL-21R.sup.-/- mice (mice per group: N=27 for week
9, N=19 for week 12). (FIG. 20D) Real-time PCR analysis of Th2
inflammatory genes in granulomatous liver tissue (N=8-10 per
group/time point). Data shown are the combined results of three
separate experiments conducted on week 9 and two performed on week
12. The asterisks denote significant differences from wild-type
values at the given time point, * p<0.05, ** p<0.01, ***
p<0.001.
[0043] FIG. 21 shows that the cellular composition of granulomas is
unchanged by IL-21R deficiency. (FIG. 21A) The cellular composition
of granulomas was evaluated in the livers of 9 week infected WT
(N=10) and IL-21R.sup.-/- (N=9) mice. The average .+-.SEM of small
lymphocytes (Sm Lym), large lymphocytes (Lg Lym), macrophages
(Mac), Fibroblasts (Fibro), Eosinophils (Eos), and Mast Cells (Mc)
are shown. (FIG. 21B) Lymphocytes were isolated from the perfused
lungs of naive WT and IL-21R.sup.-/- mice (top panels) and on day 7
following i.v. challenge with 5000 S. mansoni eggs (bottom panels).
The numbers in the histograms indicate the percentages of CD4.sup.-
and CD4.sup.+ T cells among total lung lymphocytes.
[0044] FIG. 22 shows that IL-21R-deficiency significantly slows the
progression of T.sub.H2 cytokine-dependent fibrosis. WT (open bars)
and IL-21R.sup.-/- mice (filled bars) were infected with S. mansoni
cercariae. Animals were sacrificed at 9 (acute), 12 (chronic)
(panels A-D) or 29 weeks (late chronic) (panel E) post-infection.
(FIG. 22A) The average worm pairs, total worms and eggs/worm pair
in thousands .+-.SE are shown for each group. No difference in
infection intensity was noted in any experiment (n=number of mice).
(FIG. 22B) Mice were bled at the time of sacrifice and SEA
isotype-specific Ab titers were determined by ELISA. (FIG. 22C)
Total serum IgE values in .mu.g/ml. (FIGS. 22D-F) Fibrosis was
assessed by the amount of hydroxyproline in micromoles detected in
the liver per 10,000 eggs (FIG. 22D) or in total liver (FIGS. 22E
and F). In FIG. 22F, infected WT C57BL/6 mice were treated with
either an IgG2a control antibody (cIg--open bar) or with sIL-21R-Fc
(filled bar) for 6 weeks. The asterisks denote significant
differences from wild-type values at the given time point, *
p<0.05, ** p<0.01, *** p<0.001.
[0045] FIG. 23 shows that IL-21 signaling promotes alternative
macrophage activation by modulating IL-13 receptor expression. Bone
marrow-derived macrophages were treated with various combinations
of IL-4 (20 ng/ml), IL-13 (20 ng/ml), and IL-21 (20 ng/ml)
overnight. Macrophages treated with IL-4, IL-13, and IL-21 were
pretreated with IL-21 for 6 hours prior to administration of IL-4
and IL-13. Cells were lysed 20 hr later and RNA was analyzed
individually by real-time RT-PCR. (FIG. 23A) The ability of IL-21
to promote alternative macrophage activation was assessed by
measuring Arg-1 and FIZZ1 gene expression. (FIG. 23B) Arginase
activity was quantified in cell lysates by measuring the conversion
of L-arginine to urea (mg/dL.+-.SEM, triplicate measurements) (FIG.
23C) Expression of IL-4R.alpha. and IL-13R.alpha.1 mRNA was
evaluated by real-time PCR. IL-13R.alpha.2 mRNA was nearly
undetectable in all conditions (not shown). The data shown in FIGS.
23A-C are representative of three separate experiments. (FIG. 23D)
Naive C57BL/6 mice were challenged intravenously with 5000 live S.
mansoni eggs and treated with PBS or rIL-21 (2 .mu.g/dose) every
other day from day 1 through day 6. Animals (five per group) were
sacrificed on day 7 and lung IL-13R.alpha.2 mRNA levels were
assayed by real-time PCR and expressed as fold-increase over
untreated controls (open bar). Mice were also bled at the time of
sacrifice and the amount of sIL-13R.alpha.2 in individual serum
samples was assayed by ELISA. The asterisks denote significant
differences, * p<0.05, ** p<0.01, *** p<0.001.
[0046] FIG. 24 shows that alternatively activated macrophages do
not produce significant quantities of active TGF-.beta.1. The right
panel shows active TGF-.beta.1 following macrophage activation,
while the left panel shows total TGF-.beta.1 levels following
macrophage activation. Bone marrow-derived macrophages were treated
with various combinations of IL-4 (20 ng/ml), IL-13 (20 ng/ml), and
IL-21 (20 ng/ml) overnight. Macrophages treated with IL-4, IL-13,
and IL-21 were pretreated with IL-21 for 6 hours prior to
administration of IL-4 and IL-13. 20 hours post-activation,
supernatants were assayed for total and active TGF-.beta.1 by
ELISA. High levels of total TGF-.beta.1 were detected in all groups
(e.g., compare left panel "IL-4" with "Untreated") except for cells
treated with IL-21 alone (compare left panel "IL-21" with
"Untreated"). Although total TGF-.beta.1 expression was high,
active TGF-.beta.1 was minimal in all groups (right panel). The
data shown are representative of three separate experiments
producing similar results.
DETAILED DESCRIPTION OF THE INVENTION
[0047] To examine the role of the IL-21/IL-21R signaling pathway in
the pathogenesis of fibrosis, immune responses were compared in
mice lacking a functional IL-21R (IL-21R.sup.-/-) and in wild type
mice using various models of lung and liver inflammation. In one
model, live schistosome eggs were injected intravenously into naive
or antigen-sensitized animals to study primary and secondary
granulomatous inflammation in the lung. In another model, mice were
infected percutaneously with S. mansoni cercariae and the
development of egg-induced inflammation and fibrosis was observed
in the liver. In another model, mice were infected with N.
brasiliensis. Using these models, the influence of the IL-21R on
type-2 cytokine-driven pathology in acute and chronic disease
settings was studied. The results demonstrate an important role for
the IL-21R in the generation of polarized type-2 responses in vivo,
particularly in type-2 cytokine-mediated inflammation and
fibrosis.
[0048] The present invention therefore provides methods for
treating, ameliorating, or preventing fibrosis or
fibrosis-associated disorders in a subject (e.g., a human, e.g., a
human patient) using an agent(s) that reduces the level of IL-21
and/or IL-21R (e.g., the level of expression of IL-21 and/or IL-21R
(e.g., the level of IL-21 and/or IL-21R gene products (i.e.,
protein and/or mRNA)), the level of activity of IL-21 and/or
IL-21R, the level of interaction of IL-21 with IL-21R, etc.)
relative to an untreated control (e.g., a control subject afflicted
with fibrosis or a fibrosis-associated condition, a control subject
not afflicted with fibrosis or a fibrosis-associated condition) or
relative to an appropriate reference level. In relation to
identifying an agent(s) that reduces the level of IL-21 and/or
IL-21R, measuring "the level of IL-21 and/or IL-21R" includes, but
is not limited to, (1) measuring the level of expression of IL-21
and/or IL-21R (e.g., measuring the level of IL-21 and/or IL-21R
gene products (e.g., protein and/or its corresponding mRNA)); (2)
measuring the level of activity of IL-21 and/or IL-21R; and (3)
measuring the level of interaction of IL-21 with IL-21R (e.g., in a
cell or sample of interest, e.g., from a subject (e.g., a human
patient, a control subject)). As described in further detail
herein, exemplary agents useful to treat, ameliorate, and/or
prevent fibrosis or fibrosis-associated conditions or disorders
include anti-IL-21R antibodies, antigen-binding fragments of
anti-IL-21R antibodies, anti-IL-21 antibodies, antigen-binding
fragments of anti-IL-21 antibodies, and soluble fragments of IL-21R
polypeptides. The invention further provides methods for monitoring
the course of treatment of fibrosis or a fibrosis-associated
disorder, diagnosing and prognosing the same, and screening for
compounds useful to treat fibrosis or a fibrosis-associated
disorder.
[0049] As used herein, "IL-21" or "IL-21R" means any polypeptide
that is substantially identical to the naturally occurring IL-21 or
IL-21 receptor protein, respectively. The nucleotide and amino acid
sequences encoding human interleukin-21 (IL-21) and its receptor
(IL-21R) are described, for example, in WO 00/53761, WO 01/85792,
Parrish-Novak et al. (2000) Nature 408:57-63, and Ozaki et al.
(2000) Proc. Natl. Acad. Sci. U.S.A. 97:11439-44. Desirably, the
IL-21 polypeptide binds IL-21R or the IL-21R polypeptide binds
IL-21, and upon interaction there is an increase in the signaling
activity of the IL-21/IL-21R pathway by at least 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or more than 100% above control
levels, as measured by any standard method. IL-21R is also known as
"MU-1," "NILR," and "zalpha11."
[0050] An agent that decreases the level of IL-21 and/or IL-21R
encompasses any agent that decreases the signaling activity of the
IL-21/IL-21R pathway, the level of activity of IL-21 and/or IL-21R,
the level of expression of IL-21 and/or IL-21R, and/or the level of
interaction of IL-21 and IL-21R by at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100%. Optionally, the decrease in
levels of IL-21 and/or IL-21R is assessed by measuring the level of
reduction in fibrosis. An agent that decreases the level of
interaction of IL-21 with IL-21R encompasses any agent that
decreases the interaction of IL-21 with IL-21R by at least 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. These
aforementioned agents that decrease the level of activity or
expression of IL-21 and/or IL-21R and/or decrease the level of
interaction of IL-21 with IL-21R (i.e., agents that decrease the
level of IL-21 and/or IL-21R) may be referred to herein as
"antagonists" of IL-21 and/or IL-21R.
[0051] An "IL-21 gene" or "IL-21R gene" is defined as a nucleic
acid that encodes an IL-21 or IL-21R polypeptide, respectively.
[0052] "Fibrosis" is defined as any pathological condition
resulting from an overproduction or aberrant production of fibrous
tissue. Fibrosis may occur in any organ including, for example,
kidney, lung, liver, skin, central nervous system, bone, bone
marrow, cardiovascular system, an endocrine organ or the
gastrointestinal system. By "fibrosis-associated condition" is
meant any condition that is related to fibrosis. Thus,
fibrosis-associated conditions may be caused by, be concomitant
with, or cause fibrosis.
[0053] Decreasing the level of activity of IL-21 and/or IL-21R may
refer to a reduction in the level or biological activity of IL-21
relative to the level or biological activity of IL-21 and/or IL-21R
in an untreated control or reference sample (e.g., a reference
level). Such level or activity may be decreased by at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. A decrease in the
activity of IL-21 and/or IL-21R may also be associated with a
reduction in type-2 ("Th2") cytokine expression and/or function,
which may include a modulation in, e.g., IL-4, IL-13, AMCase, Ym1,
and Fizz1/RELM.alpha.-levels and activity.
[0054] Decreasing the level of interaction of IL-21 with IL-21R may
refer to a decrease in the interaction in a treated cell or sample
relative to the level of interaction of IL-21 with IL-21R in an
untreated control or reference sample. Such level of interaction
may be decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 100%. The level of interaction may be assessed by
several well-known molecular biology techniques, e.g., ELISA and
Western blotting.
[0055] Decreasing the level IL-21 and/or IL-21R gene products
refers to a decrease in the mRNA and/or protein expression level in
a treated cell or sample relative to the gene or protein expression
level of IL-21 and/or IL-21R in an untreated control or reference
sample. Such expression may be decreased by at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of expression may
be assessed by a number of well-known molecular biology techniques,
e.g., Northern blotting or Western blotting.
[0056] By "treating or ameliorating fibrosis" is meant decreasing
the level of fibrosis relative to an untreated control, as measured
by any standard method. A reduction in fibrosis may also be
measured by a reduction in any symptom associated with fibrosis or
a fibrosis-associated condition. The examples disclosed herein
provide exemplary methods of determining whether the level of
fibrosis is decreased relative to a control.
[0057] By "treating or ameliorating a fibrosis-associated condition
(or disorder)" is meant decreasing such condition before or after
it has occurred. As compared with an equivalent untreated control,
such reduction or degree of prevention is at least 5%, 10%, 20%,
40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard
technique. A subject who is being treated for a fibrosis-associated
condition is one who a medical practitioner has diagnosed as having
such a condition. Diagnosis may be by any suitable means. A subject
in whom the development of a fibrosis-associated condition is being
prevented may or may not have received such a diagnosis. One in the
art will understand that these subjects (e.g., patients) may have
been subjected to standard tests for diagnosing fibrosis-associated
conditions or may have been identified, without examination, as one
at high risk due to the presence of one or more risk factors. The
examples disclosed herein provide exemplary methods of determining
whether the level of a fibrosis-associated disorder is decreased
relative to a control.
[0058] "Preventing" refers to delaying the onset of a fibrosis or a
fibrosis-associated condition, or prohibiting the onset of fibrosis
or a fibrosis-associated condition in a subject likely to develop
such a condition.
[0059] By "an effective amount" is meant an amount of a compound,
alone or in a combination, required to treat, ameliorate, reduce or
prevent fibrosis or a fibrosis-associated condition in a mammal.
The effective amount of active compound(s) varies depending upon
the route of administration, age, body weight, and general health
of the subject. Ultimately, the attending physician or veterinarian
will decide the appropriate amount and dosage regimen.
[0060] By "substantially identical," when referring to a protein or
polypeptide, is meant a protein or polypeptide exhibiting at least
75%, but preferably 85%, more preferably 90%, most preferably 95%,
or even 99% identity to a reference amino acid sequence, e.g., SEQ
ID NO:2, SEQ ID NO:5, and fusion proteins such as those set forth
in SEQ ID NOs:11, 13, 15, 17, 19, 21, 23, 25 and 27. For proteins
or polypeptides, the length of comparison sequences will generally
be at least 20 amino acids, preferably at least 30 amino acids,
more preferably at least 40 amino acids, and most preferably 50
amino acids or the full-length protein or polypeptide. Nucleic
acids that encode such "substantially identical" proteins or
polypeptides constitute examples of "substantially identical"
nucleic acids. It is recognized that, due to the redundancy of the
genetic code, several nucleic acids may encode a given protein or
polypeptide; such nucleic acids are within the scope of the
invention if they encode a polypeptide that is "substantially
identical" to a reference polypeptide.
[0061] The nucleic acids related to the present invention may
comprise DNA or RNA and may be wholly or partially synthetic.
Reference to a nucleotide sequence as set forth herein encompasses
a DNA molecule with the specified sequence (or a complement
thereof), and encompasses an RNA molecule with the specified
sequence in which U is substituted for T, unless context requires
otherwise.
[0062] The isolated polynucleotides related to the present
invention may be used as hybridization probes and primers to
identify and isolate nucleic acids having sequences identical to or
similar to those encoding the disclosed polynucleotides.
Hybridization methods for identifying and isolating nucleic acids
include polymerase chain reaction (PCR), Southern hybridization, in
situ hybridization and Northern hybridization, and are well known
to those skilled in the art.
[0063] Hybridization reactions may be performed under conditions of
different stringency. The stringency of a hybridization reaction
includes the difficulty with which any two nucleic acid molecules
will hybridize to one another. Preferably, each hybridizing
polynucleotide hybridizes to its corresponding polynucleotide under
reduced stringency conditions, more preferably stringent
conditions, and most preferably highly stringent conditions.
Examples of stringency conditions are shown in Table 1 below:
highly stringent conditions are those that are at least as
stringent as, for example, conditions A-F; stringent conditions are
at least as stringent as, for example, conditions G-L; and reduced
stringency conditions are at least as stringent as, for example,
conditions M-R.
TABLE-US-00001 TABLE 1 Stringency Conditions Poly- Hybrid Wash
Stringency nucleotide Length Hybridization Temperature and
Temperature and Condition Hybrid (bp).sup.1 Buffer.sup.2
Buffer.sup.2 A DNA:DNA >50 65.degree. C.; 1xSSC -or- 65.degree.
C.; 0.3xSSC 42.degree. C.; 1xSSC, 50% formamide B DNA:DNA <50
T.sub.B*; 1xSSC T.sub.B*; 1xSSC C DNA:RNA >50 67.degree. C.;
1xSSC -or- 67.degree. C.; 0.3xSSC 45.degree. C.; 1xSSC, 50%
formamide D DNA:RNA <50 T.sub.D*; 1xSSC T.sub.D*; 1xSSC E
RNA:RNA >50 70.degree. C.; 1xSSC -or- 70.degree. C.; 0.3xSSC
50.degree. C.; 1xSSC, 50% formamide F RNA:RNA <50 T.sub.F*;
1xSSC T.sub.F*; 1xSSC G DNA:DNA >50 65.degree. C.; 4xSSC -or-
65.degree. C.; 1xSSC 42.degree. C.; 4xSSC, 50% formamide H DNA:DNA
<50 T.sub.H*; 4xSSC T.sub.H*; 4xSSC I DNA:RNA >50 67.degree.
C.; 4xSSC -or- 67.degree. C.; 1xSSC 45.degree. C.; 4xSSC, 50%
formamide J DNA:RNA <50 T.sub.J*; 4xSSC T.sub.J*; 4xSSC K
RNA:RNA >50 70.degree. C.; 4xSSC -or- 67.degree. C.; 1xSSC
50.degree. C.; 4xSSC, 50% formamide L RNA:RNA <50 T.sub.L*;
2xSSC T.sub.L*; 2xSSC M DNA:DNA >50 50.degree. C.; 4xSSC -or-
50.degree. C.; 2xSSC 40.degree. C.; 6xSSC, 50% formamide N DNA:DNA
<50 T.sub.N*; 6xSSC T.sub.N*; 6xSSC O DNA:RNA >50 55.degree.
C.; 4xSSC -or- 55.degree. C.; 2xSSC 42.degree. C.; 6xSSC, 50%
formamide P DNA:RNA <50 T.sub.P*; 6xSSC T.sub.P*; 6xSSC Q
RNA:RNA >50 60.degree. C.; 4xSSC -or- 60.degree. C.; 2xSSC
45.degree. C.; 6xSSC, 50% formamide R RNA:RNA <50 T.sub.R*;
4xSSC T.sub.R*; 4xSSC .sup.1The hybrid length is that anticipated
for the hybridized region(s) of the hybridizing polynucleotides.
When hybridizing a polynucleotide to a target polynucleotide of
unknown sequence, the hybrid length is assumed to be that of the
hybridizing polynucleotide. When polynucleotides of known sequence
are hybridized, the hybrid length can be determined by aligning the
sequences of the polynucleotides and identifying the region or
regions of optimal sequence complementarity. .sup.2SSPE (1xSSPE is
0.15M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can
be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium
citrate) in the hybridization and wash buffers; washes are
performed for 15 minutes after hybridization is complete. T.sub.B*
- T.sub.R*: The hybridization temperature for hybrids anticipated
to be less than 50 base pairs in length should be 5-10.degree. C.
less than the melting temperature (T.sub.m) of the hybrid, where
T.sub.m is determined according to the following equations. For
hybrids less than 18 base pairs in length, T.sub.m(.degree. C.) =
2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18
and 49 base pairs in length, T.sub.m(.degree. C.) = 81.5 +
16.6(log.sub.10Na.sup.+) + 0.41(% G + C) - (600/N), where N is the
number of bases in the hybrid, and Na.sup.+ is the concentration of
sodium ions in the hybridization buffer (Na.sup.+ for 1xSSC =
0.165M). Additional examples of stringency conditions for
polynucleotide hybridization are provided in Sambrook, J., E. F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, chapters 9 and 11, and Current Protocols in Molecular Biology,
1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc.,
sections 2.10 and 6.3-6.4, incorporated herein by reference.
[0064] The isolated polynucleotides related to the present
invention may be used as hybridization probes and primers to
identify and isolate DNA having sequences encoding allelic variants
of the disclosed polynucleotides. Allelic variants are naturally
occurring alternative forms of the disclosed polynucleotides that
encode polypeptides that are identical to or have significant
similarity to the polypeptides encoded by the disclosed
polynucleotides. Preferably, allelic variants have at least 90%
sequence identity (more preferably, at least 95% identity; most
preferably, at least 99% identity) with the disclosed
polynucleotides. Alternatively, significant similarity exists when
the nucleic acid segments will hybridize under selective
hybridization conditions (e.g., highly stringent hybridization
conditions) to the disclosed polynucleotides.
[0065] The isolated polynucleotides related to the present
invention may also be used as hybridization probes and primers to
identify and isolate DNAs having sequences encoding polypeptides
homologous to the disclosed polynucleotides. These homologs are
polynucleotides and polypeptides isolated from a different species
than that of the disclosed polypeptides and polynucleotides, or
within the same species, but with significant sequence similarity
to the disclosed polynucleotides and polypeptides. Preferably,
polynucleotide homologs have at least 50% sequence identity (more
preferably, at least 75% identity; most preferably, at least 90%
identity) with the disclosed polynucleotides, whereas polypeptide
homologs have at least 30% sequence identity (more preferably, at
least 45% identity; most preferably, at least 60% identity) with
the disclosed polypeptides. Preferably, homologs of the disclosed
polynucleotides and polypeptides are those isolated from mammalian
species.
[0066] Calculations of "homology" or "sequence identity" between
two sequences are performed by means well known to those of skill
in the art. For example, one general means for calculating sequence
identity is described as follows. The sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in one or
both of a first and a second amino acid or nucleic acid sequence
for optimal alignment and nonhomologous sequences can be
disregarded for comparison purposes). In a preferred embodiment,
the length of a reference sequence aligned for comparison purposes
is at least 30%, preferably at least 40%, more preferably at least
50%, even more preferably at least 60%, and even more preferably at
least 70%, 80%, 90%, 100% of the length of the reference sequence.
The amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[0067] The comparison of sequences and determination of percent
sequence identity between two sequences may be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-53) algorithm,
which has been incorporated into the GAP program in the GCG
software package (available at www.gcg.com), using either a Blossum
62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,
8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet
another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at www.gcg.com), using a NWSgapdna.CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3, 4, 5, or 6. One preferred set of parameters is a
Blossum 62 scoring matrix with a gap penalty of 12, a gap extend
penalty of 4, and a frameshift gap penalty of 5. The percent
identity between two amino acid or nucleotide sequences can also be
determined using the algorithm of Meyers and Miller ((1989) CABIOS
4:11-17), which has been incorporated into the ALIGN program
(version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4.
[0068] "Substantially pure" is defined as a nucleic acid,
polypeptide, or other molecule that has been separated from the
components that naturally accompany it, e.g., genetic material,
associated proteins, membranes, and cell debris. Typically, a
polypeptide is substantially pure if it is at least 60%, 70%, 80%,
90%, 95%, or even 99%, by weight, free from the proteins and
naturally occurring organic molecules with which it naturally
associates. For example, a substantially pure polypeptide may be
obtained by extraction from a natural source, by expression of a
recombinant nucleic acid in a cell that does not normally express
that protein, or by chemical synthesis.
[0069] The term "isolated DNA" is defined as DNA that is relatively
or substantially free of the genes and other DNA sequences that
flank the DNA in the naturally occurring genome of the organism
from which the given DNA is derived. Thus, the term "isolated DNA"
encompasses, for example, cDNA, cloned genomic DNA, and synthetic
DNA.
[0070] "IL-21 fusion" polypeptide or protein, or "IL-21R fusion"
polypeptide or protein is defined as all or part of an IL-21 or
IL-21R amino acid sequence, e.g., the IL-21R extracellular fragment
from amino acids 1-235 of SEQ ID NO:2, linked to a second,
heterologous amino acid sequence. In one embodiment, the second,
heterologous amino acid sequence is a tag sequence. Common tag
sequences include myc tags, his tags, flag tags, etc. In another
embodiment of the invention, the second, heterologous amino acid
sequence is an immunoglobulin sequence, e.g., an Fc fragment. Such
fusion proteins and polypeptides, which are described in greater
detail herein, are encoded by nucleic acid sequences referred to as
"IL-21 fusion genes" or "IL-21R fusion genes."
[0071] In the screening methods a "compound" refers to a chemical,
whether naturally occurring or artificially derived. Such compounds
may include, for example, peptides, polypeptides, synthetic organic
molecules, naturally occurring organic molecules, nucleic acid
molecules, peptide nucleic acid molecules, and components and
derivatives thereof. For example, a useful compound according to
the present invention reduces binding of IL-21 to IL-21R.
[0072] Antagonists of IL-21 and IL-21R for use in treating,
ameliorating, or preventing fibrosis or a fibrosis-associated
condition may also consist of small molecules. The term "small
molecule" refers to compounds that are not macromolecules (see,
e.g., Karp (2000) Bioinformatics Ontology 16:269-85; Verkman (2004)
AJP-Cell Physiol. 286:465-74). Thus, small molecules are often
considered those compounds that are, e.g., less than one thousand
daltons (e.g., Voet and Voet, Biochemistry, 2.sup.nd ed., ed. N.
Rose, Wiley and Sons, New York, 14 (1995)). For example, Davis et
al. (2005) Proc. Natl. Acad. Sci. USA 102:5981-86, use the phrase
small molecule to indicate folates, methotrexate, and
neuropeptides, while Halpin and Harbury (2004) PLos Biology
2:1022-30, use the phrase to indicate small molecule gene products,
e.g., DNAs, RNAs and peptides. Examples of natural and synthesized
small molecules include, but are not limited to, cholesterols,
neurotransmitters, siRNAs, and various chemicals listed in numerous
commercially available small molecule databases, e.g., FCD (Fine
Chemicals Database), SMID (Small Molecule Interaction Database),
ChEBI (Chemical Entities of Biological Interest), and CSD
(Cambridge Structural Database) (see, e.g., Alfarano et al. (2005)
Nuc. Acids Res. Database Issue 33:D416-24).
[0073] The term "pharmaceutical composition" means any composition
that contains at least one therapeutically or biologically active
agent and is suitable for administration to a subject. Any of these
formulations can be prepared by well-known and accepted methods of
the art. See, for example, Remington: The Science and Practice of
Pharmacy, 21.sup.st Ed., (ed. A. R. Gennaro), Lippincott Williams
& Wilkins, Baltimore, Md. (2005).
[0074] The present invention provides significant advantages over
standard therapies for treatment and prevention of
fibrosis-associated conditions. As described herein, administration
of a therapeutic agent that reduces the level of activity or
expression of IL-21 and/or IL-21R or decreases the interactions
between IL-21 and IL-21R (i.e., reduces the level of IL-21 and/or
IL-21R activity) results in amelioration, reduction or prevention
of fibrosis and fibrosis-associated conditions. In addition, the
compound screening methods, provided by this invention, allow one
to identify novel therapeutics that modify the injury process,
rather than merely mitigating the symptoms.
Fibrotic Disorders
[0075] The generation of granulation tissue is a carefully
orchestrated process in which the expression of protease inhibitors
and extracellular matrix proteins is upregulated and the expression
of proteases is reduced, leading to the accumulation of
extracellular matrix. Abnormal accumulation of fibrous materials,
however, may ultimately lead to organ failure (e.g., Border et al.
(1994) New Engl. J. Med. 331:1286-92). The development of fibrotic
conditions, whether induced or spontaneous, is caused at least in
part by the stimulation of fibroblast activity. The influx of
inflammatory cells and activated fibroblasts into the injured organ
depends on the ability of these cell types to interact with the
interstitial matrix, which contains primarily collagens. Exemplary
tissues that may be affected by fibrosis include the kidney, lung,
liver, skin, central nervous system, bone, bone marrow, tissues of
the cardiovascular system, endocrine organs, and tissues of the
gastrointestinal system.
[0076] The methods and compositions of the present invention are
useful for any fibrosis or fibrosis-associated condition affecting
any tissue including, for example, fibrosis of an internal organ, a
cutaneous or dermal fibrosing disorder, and fibrotic conditions of
the eye. Fibrosis of internal organs (e.g., liver, lung, kidney,
heart blood vessels, gastrointestinal tract) occurs in disorders
such as pulmonary fibrosis, idiopathic fibrosis, autoimmune
fibrosis, myelofibrosis, liver cirrhosis, veno-occlusive disease,
mesangial proliferative glomerulonephritis, crescentic
glomerulonephritis, diabetic nephropathy, renal interstitial
fibrosis, renal fibrosis in subjects receiving cyclosporin,
allograft rejection, HIV associated nephropathy. Other
fibrosis-associated disorders include systemic sclerosis,
eosinophilia-myalgia syndrome, and fibrosis-associated CNS
disorders such as intraocular fibrosis. Dermal fibrosing disorders
include, for example, scleroderma, morphea, keloids, hypertrophic
scars, familial cutaneous collagenoma, and connective tissue nevi
of the collagen type. Fibrotic conditions of the eye include
conditions such as diabetic retinopathy, post-surgical scarring
(for example, after glaucoma filtering surgery and after
crossed-eyes (strabismus) surgery), and proliferative
vitreoretinopathy. Additional fibrotic conditions that may be
treated by the methods of the present invention may result, for
example, from rheumatoid arthritis, diseases associated with
prolonged joint pain and deteriorated joints; progressive systemic
sclerosis, polymyositis, dermatomyositis, eosinophilic fasciitis,
morphea, Raynaud's syndrome, and nasal polyposis. As described
herein, an IL-21/IL-21R pathway antagonist may be administered to
treat or prevent fibrosis and fibrosis-associated disorders, or to
ameliorate one or more of the symptoms associated with these
disorders.
IL-21 or IL-21R Antagonists (IL-21/IL-21R Antagonists)
[0077] The IL-21 antagonists or IL-21R antagonists of the invention
interact with IL-21 or IL-21R (e.g., mammalian IL-21 or IL-21R such
as human, bovine, rat, mouse, horse, or dog), respectively, and
reduce the level of IL-21 and/or IL-21R, e.g., reduce one or more
biological activities associated with IL-21 and/or IL-21R. When
this interaction involves direct binding, antagonists bind to IL-21
or IL-21R with high affinity (e.g., with an affinity constant of at
least about 10.sup.7 M.sup.-1, preferably about 10.sup.8 M.sup.-1,
and more preferably, about 10.sup.9 M.sup.-1 to 10.sup.10 M.sup.-1
or stronger).
[0078] The level of IL-21 and/or IL-21R is desirably reduced by
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. An
antagonist may, for example, reduce the activity of IL-21R by
neutralizing IL-21. An antagonist may be a fusion protein that
includes a fragment of an IL-21R fused to a non-IL-21R fragment
such as an immunoglobulin Fc region, e.g., the fusion proteins set
forth in SEQ ID NOs:11, 13, 15, 17, 19, 21, 23, 25 and 27. Other
exemplary antagonists are anti-IL-21R or anti-IL-21 antibodies or
antigen-binding fragments thereof, soluble forms of IL-21R,
peptides, inhibitory polynucleotides (e.g., siRNAs, SNPs, and
aptamers) and small molecules.
[0079] In one embodiment, the IL-21/IL-21R antagonist is an
anti-IL-21R or anti-IL-21 antibody, or an antigen-binding fragment
thereof. In a further embodiment, the antibody is a neutralizing
antibody. If desired, the antibody may be a monoclonal or
single-specificity antibody that binds to IL-21 or IL-21R or an
antigen-binding fragment thereof (e.g., an Fab, F(ab').sub.2, Fv or
a single chain Fv fragment). The antibody may be human, humanized,
chimeric, or in vitro-generated antibody to IL-21 or IL-21R
polypeptide.
[0080] Alternatively, the IL-21 antagonist or IL-21R antagonist may
be a full length (e.g., a mutated sequence) or a fragment of an
IL-21 polypeptide or an IL-21R polypeptide (e.g., human). Exemplary
antagonists include, for example, an inhibitory IL-21
receptor-binding domain of an IL-21 polypeptide (e.g., human) or
the extracellular domain of murine or human IL-21R. The IL-21
antagonist may have an amino acid sequence that is substantially
identical to (e.g., having at least 85%, 90%, 95%, 98%, 99%
sequence identity with) the naturally occurring IL-21R (e.g., SEQ
ID NO:2 (human) or SEQ ID NO:5 (murine)) or a fragment thereof (see
Table 2). Alternatively, the antagonist may have an amino acid
sequence encoded by a nucleotide sequence that is substantially
identical to the naturally occurring mammalian IL-21R or a fragment
thereof (e.g., SEQ ID NO:1 (human) or SEQ ID NO:4 (murine)) or by a
nucleotide sequence that hybridizes to one of the foregoing
nucleotide sequences under stringent conditions, e.g., highly
stringent conditions (see Table 1).
TABLE-US-00002 TABLE 2 Summary of Sequences Designation Description
SEQ ID NO: 1 Nucleic acid sequence of human IL-21R cDNA SEQ ID NO:
2 Amino acid sequence of human IL-21R SEQ ID NO: 3 Amino acid
sequence of conserved WSXWS motif SEQ ID NO: 4 Nucleic acid
sequence of mouse IL-21R cDNA SEQ ID NO: 5 Amino acid sequence of
mouse IL-21R SEQ ID NO: 6 Amino acid sequence of Fc fragment SEQ ID
NO: 7 Nucleic acid (cDNA) sequence of human IL-21 SEQ ID NO: 8
Amino acid sequence of human IL-21 SEQ ID NO: 9 Peptide signal
sequence SEQ ID NO: 10 Nucleic acid (cDNA) sequences of human
IL-21R monomer fused to a honeybee leader sequence and amino
terminal His.sub.6 and Flag tags SEQ ID NO: 11 Amino acid sequence
of human IL-21R monomer (20-235) fused to a honeybee leader
sequence and amino terminal His.sub.6 and Flag tags SEQ ID NO: 12
Nucleic acid (cDNA) sequence of human IL-21R extracellular domain
(1-235) fused to an IgG fragment SEQ ID NO: 13 Amino acid sequence
of human IL-21R extracellular domain (1-235) fused to an IgG
fragment SEQ ID NO: 14 Nucleic acid (cDNA) sequence of human IL-21R
extracellular domain (1-235) fused to an IgG fragment and a
His.sub.6 tag SEQ ID NO: 15 Amino acid sequence of human IL-21R
extracellular domain (1-235) fused to an IgG fragment and a
His.sub.6 tag SEQ ID NO: 16 Nucleic acid (cDNA) sequence of human
IL-21R extracellular domain (1-235) fused to an IgG fragment
mutated at residues 254 and 257 SEQ ID NO: 17 Amino acid sequence
of human IL-21R extracellular domain (1-235) fused to an IgG
fragment mutated at residues 254 and 257 SEQ ID NO: 18 Nucleic acid
(cDNA) sequence of human IL-21R monomer fused to a Rhodopsin tag
SEQ ID NO: 19 Amino acid sequence of human IL-21R monomer (20-235)
fused to a Rhodopsin tag SEQ ID NO: 20 Nucleic acid (cDNA) sequence
of human IL-21R extracellular domain (1-235) fused to EK cleavage
sites and an IgG1 fragment with a mutated Fc region SEQ ID NO: 21
Amino acid sequence of human IL-21R extracellular domain (1-235)
fused to EK cleavage sites and an IgG1 fragment with a mutated Fc
region SEQ ID NO: 22 Nucleic acid sequence of mouse IL-21R
extracellular domain fused to a mouse genomic IgG2a fragment SEQ ID
NO: 23 Amino acid sequence of mouse IL-21R extracellular domain
fused to a mouse genomic IgG2a fragment SEQ ID NO: 24 Nucleic acid
(genomic) sequence of mouse IL-21R extracellular domain fused to
Flag and His.sub.6 tags SEQ ID NO: 25 Amino acid sequence of mouse
IL-21R extracellular domain fused to fused to Flag and His.sub.6
tags SEQ ID NO: 26 Nucleic acid sequence of mouse IL-21R
extracellular domain fused to a honeybee leader sequence and amino
terminal Flag and His.sub.6 tags SEQ ID NO: 27 Amino acid sequence
of mouse IL-21R extracellular domain fused to a honeybee leader
sequence and amino terminal Flag and His.sub.6 tags SEQ ID NO: 28
Sense primer for mouse IL-21 SEQ ID NO: 29 Antisense primer for
mouse IL-21 SEQ ID NO: 30 Sense primer for mouse IL-21R SEQ ID NO:
31 Antisense primer for mouse IL-21R SEQ ID NO: 32 Sense primer for
mouse IFN-.gamma. SEQ ID NO: 33 Antisense primer for mouse
IFN-.gamma. SEQ ID NO: 34 Peptide linker
(Ser-Gly-Gly-Gly-Gly).sub.y, wherein "y" is 1, 2, 3, 4, 5, 6, 7, or
8 SEQ ID NO: 35 Human IL-2 beta chain
[0081] Optionally, the IL-21R polypeptide may be a soluble
polypeptide incapable of membrane anchoring. Such soluble
polypeptides include, for example, IL-21R polypeptides that lack a
sufficient portion of their membrane spanning domain or are
modified such that the membrane spanning domain is nonfunctional.
For example, the IL-21R polypeptide may be a soluble fragment of an
IL-21R (e.g., a fragment of an IL-21R containing the extracellular
domain of murine or human IL-21R, including an amino acid sequence
from about amino acids 1-235, 1-236, 20-235, or 20-236 of SEQ ID
NO:2 (human), or from about amino acids 1-236, or 20-236 of SEQ ID
NO:5 (murine)). Exemplary IL-21 antagonists may have an amino acid
sequence that is substantially identical to amino acids 20-538 of
SEQ ID NO:2 (mature human IL-21R), amino acids 1-235 of SEQ ID NO:2
(extracellular domain of human IL-21R), amino acids 1-236 of SEQ ID
NO:2, amino acids 20-235 of SEQ ID NO:2, amino acids 20-236 of SEQ
ID NO:2, amino acids 1-236 of SEQ ID NO:5, or amino acids 20-236 of
SEQ ID NO:5.
[0082] An IL-21 antagonist of the invention may also be encoded by
nucleic acids that hybridize to the nucleotide sequence set forth
in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, or SEQ ID NO:26, under highly stringent conditions, e.g.,
as set forth in Table 1. Isolated polynucleotides which encode
IL-21R proteins or fusion proteins, but which differ from the
nucleotide sequence set forth in SEQ ID NOs:1, 4, 10, 12, 14, 16,
18, 20, 22, 24, or 26, by virtue of the degeneracy of the genetic
code, are also encompassed by the present invention. Variations in
the nucleotide sequence as set forth in SEQ ID NOs:1, 4, 10, 12,
14, 16, 18, 20, 22, 24, or 26, which are caused by point mutations
or by induced modifications, are also included in the
invention.
[0083] If desired, a soluble IL-21R polypeptide may include, or be
fused to, a second moiety such as a polypeptide (e.g., an
immunoglobulin chain, a GST, Lex-A or MBP polypeptide sequence).
For example, a fusion protein may include a fragment of an IL-21R
polypeptide, which is capable of binding IL-21, such as a soluble
fragment of an IL-21R (e.g., a fragment containing the
extracellular domain of murine or human IL-21R such as amino acids
1-235, 1-236, 20-235 or 20-236 of SEQ ID NO:2 (human), or amino
acids 1-236 or 20-236 of SEQ ID NO:5 (murine)) fused to a second
moiety (e.g., an immunoglobulin chain, an Fc fragment, a heavy
chain constant regions of various immunoglobulin isotypes,
including IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and
IgE).
[0084] Desirably, the IL-21 antagonist of the invention reduces at
least one biological activity associated with the naturally
occurring IL-21R, including, for example, the ability to interact
with or bind to an IL-21 polypeptide, the ability to associate with
signal transduction molecules such as .gamma.c or JAK1, the ability
to stimulate phosphorylation and/or activation of stat proteins
(e.g., Stat 5 and/or Stat 3), and the ability to modulate (e.g.,
stimulate or decrease) proliferation, differentiation, effector
cell function, cytolytic activity, cytokine secretion, and/or
survival of immune cells such as T cells (CD8+ and CD4+ T cells,
including Th1 and Th2 cells), NK cells, B cells, macrophages, and
megakaryocytes.
[0085] According to the present invention, an IL-21 polypeptide is
a cytokine showing sequence homology to IL-2, IL-4 and IL-15
(Parrish-Novak et al. (2000) Nature 408:57-63). Despite low
sequence homology among interleukin cytokines, cytokines share a
common secondary motif, i.e., a "four-helix-bundle" structure that
is representative of the family. IL-21 is expressed primarily in
activated CD4+ T cells, and has been reported to have effects on
NK, B and T cells (Parrish-Novak et al. (2000) supra; Kasaian et
al. (2002) Immunity 16:559-69). IL-21 binds to IL-21R (also
referred to as "MU-1," "NILR," and "zalpha11"). Upon IL-21 binding,
activation of IL-21R leads to Stat5 and/or Stat3 signaling (Ozaki
et al. (2000) supra).
[0086] The amino acid sequences of IL-21 polypeptides are publicly
known. For example, the nucleotide sequence and amino acid sequence
of a human IL-21 is available at GenBank Acc. No. NM.sub.--021803.
The disclosed human IL-21 nucleotide sequence is presented
below:
TABLE-US-00003 (SEQ ID NO: 7) 1 gctgaagtga aaacgagacc aaggtctagc
tctactgttg gtacttatga gatccagtcc 61 tggcaacatg gagaggattg
tcatctgtct gatggtcatc ttcttgggga cactggtcca 121 caaatcaagc
tcccaaggtc aagatcgcca catgattaga atgcgtcaac ttatagatat 181
tgttgatcag ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga
241 agatgtagag acaaactgtg agtggtcagc tttttcctgc tttcagaagg
cccaactaaa 301 gtcagcaaat acaggaaaca atgaaaggat aatcaatgta
tcaattaaaa agctgaagag 361 gaaaccacct tccacaaatg cagggagaag
acagaaacac agactaacat gcccttcatg 421 tgattcttat gagaaaaaac
cacccaaaga attcctagaa agattcaaat cacttctcca 481 aaagatgatt
catcagcatc tgtcctctag aacacacgga agtgaagatt cctgaggatc 541
taacttgcag ttggacacta tgttacatac tctaatatag tagtgaaagt catttctttg
601 tattccaagt ggaggag
[0087] The amino acid sequence of the disclosed human IL-21
polypeptide is presented below:
TABLE-US-00004 (SEQ ID NO: 8)
MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQL
KNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINV
SIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKM
IHQHLSSRTHGSEDS
[0088] Thus, an IL-21 polypeptide refers to a protein that is
capable of interacting with or binding to IL-21R and having one of
the following features: (i) an amino acid sequence substantially
identical to a naturally occurring mammalian IL-21 or a fragment
thereof (e.g., SEQ ID NO:8 (human)); (ii) an amino acid sequence
which is encoded by a nucleotide sequence that is substantially
identical to a naturally occurring mammalian IL-21 nucleotide
sequence or a fragment thereof (e.g., SEQ ID NO:7 (human) or a
fragment thereof); (iii) an amino acid sequence encoded by a
nucleotide sequence degenerate to a naturally occurring IL-21
nucleotide sequence or a fragment thereof, e.g., SEQ ID NO:7
(human) or a fragment thereof; or (iv) a nucleotide sequence that
hybridizes to one of the foregoing nucleotide sequence sequences
under stringent conditions.
[0089] In all foregoing aspects of the invention, the IL-21 or
IL-21R polypeptides may be provided as a variant polypeptide having
a mutation in the naturally occurring IL-21 or IL-21R sequence
(wild type) that results in higher affinity (relative to the
nonmutated sequence) binding to IL-21R or IL-21, respectively. Such
mutations may be useful, for example, to increase resistance to
proteolysis (relative to the nonmutated sequence). Some amino acid
sequences in the disclosed sequences can be varied without
significantly modifying IL-21 or IL-21R structure or function. In
general, it is possible to replace residues that form IL-21 or
IL-21R protein tertiary structure, provided that residues that
perform a similar function are used. In other instances, the type
of residue may be completely irrelevant if an alteration occurs in
a noncritical area. Thus, the invention further includes IL-21 and
IL-21R variants that show substantial IL-21-type biological
activity. Such variants include deletions, insertions, inversions,
repeats, and type substitutions (for example, substituting one
hydrophilic residue for another, but not a strongly hydrophilic
residue for a strongly hydrophobic residue). Small changes or
"neutral" amino acid substitutions will often have little impact on
protein function. (Taylor (1986) J. Theor. Biol. 119:205-18).
Conservative substitutions may include, but are not limited to,
replacements among the aliphatic amino acids, substitutions between
amide residues, exchanges of basic residues, and replacements among
the aromatic residues. Further guidance concerning which amino acid
change is likely to be phenotypically silent (i.e., is unlikely to
significantly affect function) can be found in Bowie et al. (1990)
Science 247:1306-10 and Zvelebil et al. (1987) J. Mol. Biol.
195:957-61.
[0090] Optionally, the IL-21 or IL-21R antagonist is a fusion
protein containing the IL-21 or IL-21R polypeptides or fragments
thereof described herein fused to a second moiety such as an
immunoglobulin chain, e.g., an Fc fragment, an epitope (tag)
sequence, e.g., GST or myc, and additional well-known sequences
such as Lex-A, or MBP polypeptide sequence. If desired, the fusion
protein may include a fragment of an IL-21R polypeptide that is
capable of binding IL-21, such as a soluble fragment of an IL-21R
(e.g., a fragment of an IL-21R containing the extracellular domain
of murine or human IL-21R from about amino acids 1-235, 1-236,
20-235, or 20-236 of SEQ ID NO:2 (human), or from about amino acids
1-236, or 20-236 of SEQ ID NO:5 (murine) or a fragment identical
to, or substantially identical to, a polypeptide encoded by SEQ ID
NOs:1 or 4) fused to a second moiety (e.g., an immunoglobulin
chain, an Fc fragment, a heavy chain constant region(s) of the
various isotypes, including: IgG1, IgG2, IgG3, IgG4, IgM, IgA1,
IgA2, IgD, and IgE). Alternatively, the human Fc sequence has been
mutated at one or more amino acids (e.g., mutated at residues 254
and 257 of SEQ ID NO:16) in the naturally occurring sequence to
reduce Fc receptor binding. In other embodiments, the fusion
protein may include the extracellular domain of murine IL-21R (from
about amino acids 1-236, or 20-235 of SEQ ID NO:5 (murine)) fused
to a murine immunoglobulin Fc chain (including, but not limited to,
murine IgG, e.g., murine IgG2a or a mutated form of murine
IgG2a).
[0091] Examples of antagonistic fusion proteins that may be used in
the methods of the invention are shown in FIGS. 7-15. The fusion
protein may include an amino acid sequence substantially identical
to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27 or
an amino acid sequence encoded by a nucleotide sequence that is
substantially identical to SEQ ID NOs:10, 12, 14, 16, 18, 20, 22,
24, or 26. One exemplary fusion protein contains the human IL-21R
extracellular domain (e.g., amino acids 1-235 of SEQ ID NO:2) fused
at the C-terminus via a linker (corresponding to amino acids
236-243 of SEQ ID NO:17) to human immunoglobulin G1 (IgG1) Fc
mutated sequence (corresponding to amino acids 244-467 of SEQ ID
NO:17). The human Fc sequence has been mutated at residues 254 and
257 from the wild type sequence to reduce Fc receptor binding. The
nucleotide and amino acid sequences are shown as SEQ ID NO:16 and
SEQ ID NO:17, respectively.
[0092] The second polypeptide is preferably soluble. Optionally,
the second polypeptide enhances the half-life, (e.g., the serum
half-life) of the linked polypeptide. If desired, the second
polypeptide includes a sequence that facilitates association of the
fusion polypeptide with a second IL-21R or IL-21 polypeptide. The
second polypeptide may include at least a region of an
immunoglobulin polypeptide. Immunoglobulin fusions polypeptides are
known in the art and are described in, e.g., U.S. Pat. Nos.
5,225,538; 5,428,130; 5,514,582; 5,714,147; and 5,455,165.
[0093] Optionally, the second polypeptide is a full-length
immunoglobulin polypeptide or a fragment thereof (e.g., a heavy
chain, light chain, Fab, Fab.sub.2, Fv, or Fc).
[0094] In one example, the second polypeptide has less effector
function that the effector function of an Fc region of a wild-type
immunoglobulin heavy chain. Fc effector function includes for
example, Fc receptor binding, complement fixation and T
cell-depleting activity (see, for example, U.S. Pat. No.
6,136,310). Methods for assaying T cell-depleting activity, Fc
effector function, and antibody stability are known in the art. In
one embodiment, the second polypeptide has low or no affinity for
the Fc receptor. In an alternative embodiment, the second
polypeptide has low or no affinity for complement protein C1q.
[0095] A preferred second polypeptide sequence includes the amino
acid sequence of SEQ ID NO:6. This sequence includes an Fc region.
Underlined amino acids are those that differ from the amino acids
found in the corresponding positions of the wild-type
immunoglobulin sequence:
TABLE-US-00005 (SEQ ID NO: 6)
HTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0096] Fusion proteins may additionally include a linker
sequence(s) joining the first moiety to the second moiety. For
example, the fusion protein may include a peptide linker of about 4
to 20 amino acids, more preferably 5 to 10 amino acids in length,
and most preferably about 8 amino acids in length. The amino acids
in the peptide linker may include, e.g., Gly, Ser, Asn, Thr and
Ala. Thus, a peptide linker may consist of a Gly-Ser element. In
other embodiments, the fusion protein includes a peptide linker
having the formula (Ser-Gly-Gly-Gly-Gly).sub.y (SEQ ID NO:34),
wherein "y" is 1, 2, 3, 4, 5, 6, 7, or 8.
[0097] In other embodiments, additional amino acid sequences can be
added to the N- or C-terminus of the fusion protein to facilitate
expression, detection and/or isolation or purification. For
example, an IL-21/IL-21R fusion protein may be linked to one or
more additional moieties, e.g., GST (i.e., glutathione
S-transferase), His, FLAG, or myc tags. For example, the fusion
protein may additionally include a GST peptide in which the fusion
protein sequences are fused to the C-terminus of the GST sequences.
Such fusion proteins facilitate the purification or identification
of the IL-21R/MU-1 fusion protein. In other embodiments, additional
amino acid sequences may be added to the N- or C-terminus of the
fusion protein to facilitate expression, steric flexibility,
detection, and/or isolation or purification.
[0098] The fusion protein may also include a heterologous signal
sequence at its N-terminus. For example, the native IL-21R signal
sequence may be removed and replaced with a signal sequence from
another protein. In certain host cells (e.g., mammalian host
cells), expression and/or secretion of IL-21R may be increased
using a heterologous signal sequence. A signal peptide that can be
included in the fusion protein is MPLLLLLLLLPSPLHP (SEQ ID NO:9).
If desired, one or more amino acids can additionally be inserted
between the first polypeptide moiety comprising the IL-21R/MU-1
moiety and the second polypeptide moiety.
[0099] The IL-21/IL-21R antagonists described herein may be
derivatized or linked to another functional molecule, e.g., another
peptide or protein (e.g., an Fab' fragment). For example, the
fusion protein or an antibody, or antigen-binding portion, can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as an antibody (e.g., a bispecific or a
multispecific antibody), toxins, radioisotopes, cytotoxic or
cytostatic agents, among others.
[0100] A chimeric or fusion protein of the invention may be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or sticky-ended termini
for ligation, restriction enzyme digestion to create appropriate
termini, filling-in of sticky ends as appropriate, alkaline
phosphatase treatment to avoid undesirable ligation, and enzymatic
ligation. In another embodiment, the fusion gene can be synthesized
by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers that give rise to complementary overhangs
between two consecutive gene fragments that can subsequently be
annealed and reamplified to generate a chimeric gene sequence (see,
for example, Ausubel et al., supra). Moreover, many expression
vectors that encode a fusion moiety (e.g., an Fc region of an
immunoglobulin heavy chain) are commercially available. An
IL-21R/MU-1 or IL-21 encoding nucleic acid can be cloned into such
an expression vector such that the fusion moiety is linked in-frame
to the immunoglobulin protein. In some embodiments, IL-21R/MU-1 or
IL-21 fusion polypeptides exist as oligomers, such as dimers or
trimers. An IL-21R/MU-1 or IL-21 monomer, and/or nucleic acids
encoding an IL-21R/MU-1 or IL-21, can be constructed using methods
known in the art.
Production of Nucleic Acids
[0101] The isolated polynucleotides of the invention may be
operably linked to an expression control sequence, such as the pMT2
or pED expression vectors disclosed in Kaufman et al. (1991) Nuc.
Acids Res. 19:4485-90, in order to produce the IL-21R or IL-21
polypeptides (including fragments and fusions thereof)
recombinantly. Many suitable expression control sequences are known
in the art. General methods of expressing recombinant proteins are
also known and are exemplified in Kaufman (1990) Meth. Enzym.
185:537-66. As defined herein "operably linked" means enzymatically
or chemically ligated to form a covalent bond between the isolated
polynucleotide of the invention and the expression control sequence
in such a way that the IL-21R or IL-21 polypeptide is expressed by
a host cell that has been transformed (transfected) with the
ligated polynucleotide/expression control sequence.
[0102] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid,"
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., nonepisomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0103] The recombinant expression constructs of the invention may
carry additional sequences, such as regulatory sequences (i.e.,
sequences that regulate either vector replication, e.g., origins of
replication, transcription of the nucleic acid sequence encoding
the polypeptide (or peptide) of interest, or expression of the
encoded polypeptide), tag sequences such as histidine, and
selectable marker genes. The term "regulatory sequence" is intended
to include promoters, enhancers and any other expression control
elements (e.g., polyadenylation signals, transcription splice
sites) that control transcription, replication or translation. Such
regulatory sequences are described, for example, in Goeddel, Gene
Expression Technology: Methods in Enzymology, Academic Press, San
Diego, Calif. (1990). It will be appreciated by those skilled in
the art that the design of the expression vector, including the
selection of regulatory sequences, will depend on various factors,
including choice of the host cell and the level of protein
expression desired. Preferred regulatory sequences for expression
of proteins in mammalian host cells include viral elements that
direct high levels of protein expression, such as promoters and/or
enhancers derived from the FF-1a promoter and BGH poly A,
cytomegalovirus (CMV) (e.g., the CMV promoter/enhancer), Simian
virus 40 (SV40) (e.g., the SV40 promoter/enhancer), adenovirus
(e.g., the adenovirus major late promoter (AdMLP)), and polyoma.
Viral regulatory elements, and sequences thereof, are described in,
e.g., U.S. Pat. Nos. 5,168,062; 4,510,245; and 4,968,615.
[0104] The recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication and terminator sequences) and selectable marker genes.
The selectable marker gene facilitates selection of host cells into
which the vector has been introduced (see, e.g., U.S. Pat. Nos.
4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For
example, typically the selectable marker gene confers resistance of
the host cell transfected or transformed with the selectable marker
to compounds such as G418 (geneticin), hygromycin or methotrexate.
Preferred selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dhfr host cells with methotrexate
selection/amplification), the neo gene (for G418 selection), and
genes conferring tetracycline and/or ampicillin resistance to
bacteria.
[0105] Suitable vectors, containing appropriate regulatory
sequences, including promoter sequences, terminator sequences,
polyadenylation sequences, enhancer sequences, marker genes and
other sequences as appropriate, may be either chosen or
constructed. Inducible expression of proteins, achieved by using
vectors with inducible promoter sequences, such as
tetracycline-inducible vectors, e.g., pTet-On.TM. and pTet-Off.TM.
(Clontech, Palo Alto, Calif.), may also be used in the disclosed
method. For further details regarding expression vectors, see, for
example, Sambrook et al., supra. Many known techniques and
protocols for manipulation of nucleic acids, for example, in
preparation of nucleic acid constructs, mutagenesis, sequencing,
introduction of DNA into cells, gene expression, and analysis of
proteins, are also described in detail in Sambrook et al.,
supra.
[0106] A number of types of cells may act as suitable host cells
for expression of the IL-21R/MU-1 or IL-21 or fusion protein
thereof. Any cell type capable of expressing functional IL-21R/MU-1
or IL-21 protein or fusion thereof may be used. Suitable mammalian
host cells include, for example, monkey COS cells, Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431
cells, human Colo205 cells, 3T3 cells, CV-1 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants,
HeLa cells, mouse L cells, BHK, HL-60, U937, HaK,
C.sub.3H.sub.10T1/2, Rat2, BaF3, 32D, FDCP-1, PC12, M1x or C2C12
cells.
[0107] The IL-21R or IL-21 polypeptide or fusion protein thereof
may also be produced by operably linking the isolated
polynucleotide of the invention to suitable control sequences in
one or more insect expression vectors, and employing an insect
expression system. Materials and methods for baculovirus/Sf9
expression systems are commercially available in kit form (e.g.,
the MAXBAC.RTM. kit, Invitrogen, Carlsbad, Calif.). Soluble forms
of the polypeptides described herein may also be produced in insect
cells using appropriate isolated polynucleotides as described
above.
[0108] Alternatively, the IL-21R or IL-21 polypeptide or fusion
protein thereof may be produced in lower eukaryotes such as yeast,
or in prokaryotes such as bacteria. Suitable yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces
strains, Candida, or any yeast strain capable of expressing
heterologous proteins. Suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing heterologous proteins.
Expression in bacteria may result in formation of inclusion bodies
incorporating the recombinant protein. Thus, refolding of the
recombinant protein may be required in order to produce active or
more active material. Several methods for obtaining correctly
folded heterologous proteins from bacterial inclusion bodies are
known in the art. These methods generally involve solubilizing the
protein from the inclusion bodies, then denaturing the protein
completely using a chaotropic agent. When cysteine residues are
present in the primary amino acid sequence of the protein, it is
often necessary to accomplish the refolding in an environment that
allows correct formation of disulfide bonds (a redox system).
General methods of refolding are disclosed in Kohno (1990) Meth.
Enzym. 185:187-95, EP 0433225, and U.S. Pat. No. 5,399,677.
[0109] A protein of the invention (or a fragment or fusion thereof)
may also be expressed as a product of transgenic animals, e.g., as
a component of the milk of transgenic cows, goats, pigs, or sheep
which are characterized by somatic or germ cells containing a
polynucleotide sequence, e.g., encoding the IL-21R or IL-21 or
fusion protein thereof. Accordingly, the protein may be prepared by
growing a culture transformed host cells under culture conditions
necessary to express the desired protein. The resulting expressed
protein may then be purified from the culture medium or cell
extracts. Soluble forms of the protein may be purified from
conditioned media. Membrane-bound forms of IL-21R protein of the
invention can be purified by preparing a total membrane fraction
from the expressing cell and extracting the membranes with a
nonionic detergent such as TRITON.RTM. X-100 (EMD Biosciences, San
Diego, Calif.).
[0110] The polypeptides described herein may be purified using
methods known to those skilled in the art. For example, the protein
of the invention may be concentrated using a commercially available
protein concentration filter, for example, by using an AMICON.RTM.
or PELLICON.RTM. ultrafiltration unit (Millipore, Billerica,
Mass.). Following the concentration step, the concentrate may be
applied to a purification matrix such as a gel filtration medium.
Alternatively, an anion exchange resin may be employed, for
example, a matrix or substrate having pendant diethylaminoethyl
(DEAE) or polyethyleneimine (PEI) groups. The matrices may be
acrylamide, agarose, dextran, cellulose or other types commonly
employed in protein purification. Alternatively, a cation exchange
step may be employed. Suitable cation exchangers include various
insoluble matrices comprising sulfopropyl or carboxymethyl groups.
Sulfopropyl groups are preferred (e.g., S-SEPHAROSE.RTM. columns,
Sigma-Aldrich, St. Louis, Mo.). The purification of the IL-21R/MU-1
protein or fusion protein from culture supernatant may also include
one or more column steps over such affinity resins such as
concanavalin A-agarose, AF-HEPARIN650, heparin-TOYOPEARL.RTM. or
Cibacron blue 3GA SEPHAROSE.RTM. (Tosoh Biosciences, San Francisco,
Calif.); or by hydrophobic interaction chromatography using such
resins as phenyl ether, butyl ether, or propyl ether; or by
immunoaffinity chromatography. Finally, one or more reverse-phase
high performance liquid chromatography (RP-HPLC) steps employing
hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl
or other aliphatic groups, can be employed to further purify the
IL-21R/MU-1 or IL-21 protein. Affinity columns including antibodies
to the protein of the invention may also be used in purification in
accordance with known methods. Some or all of the foregoing
purification steps, in various combinations or with other known
methods, may also be employed to provide a substantially purified
isolated recombinant protein. Preferably, the isolated protein is
purified so that it is substantially free of other mammalian
proteins.
Production of Antibodies
[0111] The IL-21 or IL-21R polypeptides of the invention may be
used to immunize animals to obtain polyclonal and monoclonal
antibodies that specifically react with the IL-21 or IL-21R and
regulate the expression or activity of IL-21 and/or IL-21R, or
regulate the level of interaction of IL-21 with IL-21R. Such
antibodies may be obtained, for example, using the entire IL-21R or
fragments thereof as immunogens. The peptide immunogens may
additionally contain a cysteine residue at the carboxyl terminus
and be conjugated to a hapten such as keyhole limpet hemocyanin
(KLH). Additional peptide immunogens may be generated by replacing
tyrosine residues with sulfated tyrosine residues. Methods for
synthesizing such peptides are known in the art, for example, as in
Merrifield (1963) J. Amer. Chem. Soc. 85: 2149-54 and Krstenansky
and Mao (1987) FEBS Lett. 211:10-16.
[0112] Human monoclonal antibodies (mAbs) directed against IL-21 or
IL-21R may be generated using transgenic mice carrying the human
immunoglobulin genes rather than the mouse system. Splenocytes from
these transgenic mice immunized with the antigen of interest are
used to produce hybridomas that secrete human mAbs with specific
affinities for epitopes from a human protein (see, e.g., WO
91/00906, WO 91/10741, WO 92/03918, WO 92/03917, Lonberg et al.
(1994) Nature 368:856-59, Green et al. (1994) Nat. Genet. 7:13-21,
Morrison et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 81:6851-55,
and Tuaillon et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
90:3720-24).
[0113] Monoclonal antibodies may also be generated by other methods
known to those skilled in the art of recombinant DNA technology.
One exemplary method, referred to as the "combinatorial antibody
display" method, has been developed to identify and isolate
antibody fragments having a particular antigen specificity, and can
be utilized to produce monoclonal antibodies (for descriptions of
combinatorial antibody display see, e.g., Sastry et al. (1989)
Proc. Natl. Acad. Sci. U.S.A. 86:5728-32; Huse et al. (1989)
Science 246:1275-81; and Orlandi et al. (1989) Proc. Natl. Acad.
Sci. U.S.A. 86:3833-37). After immunizing an animal with an
immunogen as described above, the antibody repertoire of the
resulting B cell pool is cloned. The DNA sequence of the variable
regions of a diverse population of immunoglobulin molecules may be
obtained using a mixture of oligomer primers and PCR. For instance,
mixed oligonucleotide primers corresponding to the 5' leader
(signal peptide) sequences and/or framework 1 (FR1) sequences, as
well as primer to a conserved 3' constant region primer may be used
for PCR amplification of the heavy and light chain variable regions
from a number of murine antibodies (Larrick et al. (1991)
BioTechniques 11:152-56). A similar strategy may also been used to
amplify human heavy and light chain variable regions from human
antibodies (Larrick et al. (1991) Methods: Companion to Methods in
Enzymology 2:106-10).
[0114] Chimeric antibodies, including chimeric immunoglobulin
chains, may also be produced by recombinant DNA techniques known in
the art. For example, a gene encoding the Fc constant region of a
murine (or other species) monoclonal antibody molecule is digested
with restriction enzymes to remove the region encoding the murine
Fc, and the equivalent portion of a gene encoding a human Fc
constant region is substituted (see PCT/US86/02269; EP 184,187; EP
171,496; EP 173,494; WO 86/01533; U.S. Pat. No. 4,816,567; EP
125,023; Better et al. (1988) Science 240:1041-43; Liu et al.
(1987) Proc. Natl. Acad. Sci. U.S.A. 84:3439-43; Liu et al. (1987)
J. Immunol. 139:3521-26; Sun et al. (1987) Proc. Natl. Acad. Sci.
U.S.A. 84:214-18; Nishimura et al. (1987) Canc. Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-49; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-59).
[0115] If desired, an antibody or an immunoglobulin chain may be
humanized by methods known in the art. Humanized antibodies,
including humanized immunoglobulin chains, may be generated by
replacing sequences of the Fv variable region that are not directly
involved in antigen binding with equivalent sequences from human Fv
variable regions. General methods for generating humanized
antibodies are provided by Morrison (1985) Science 229:1202-07; Oi
et al. (1986) BioTechniques 4:214-21; and U.S. Pat. Nos. 5,585,089,
5,693,761 and 5,693,762, all of which are hereby incorporated by
reference in their entireties. Those methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of immunoglobulin Fv variable regions from at least one
of a heavy or light chain. Sources of such nucleic acid are well
known to those skilled in the art and, for example, may be obtained
from a hybridoma producing an antibody against a predetermined
target. The recombinant DNA encoding the humanized antibody, or
fragment thereof, may then be cloned into an appropriate expression
vector.
[0116] Humanized or CDR-grafted antibody molecules or
immunoglobulins may be produced by CDR grafting or CDR
substitution, wherein one, two, or all CDRs of an immunoglobulin
chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et
al. (1986) Nature 321:552-25; Verhoeyan et al. (1988) Science
239:1534-36; and Beidler et al. (1988) J. Immunol. 141:4053-60, all
of which are hereby incorporated by reference in their entireties.
U.S. Pat. No. 5,225,539 describes a CDR-grafting method that may be
used to prepare humanized antibodies of the present invention (see
also, GB 2188638A). All of the CDRs of a particular human antibody
may be replaced with at least a portion of a nonhuman CDR, or only
some of the CDRs may be replaced with nonhuman CDRs. It is only
necessary to replace the number of CDRs required for binding of the
humanized antibody to a predetermined antigen.
[0117] Monoclonal, chimeric and humanized antibodies, which have
been modified by, e.g., deleting, adding, or substituting other
portions of the antibody, e.g., the constant region, are also
within the scope of the invention. For example, an antibody may be
modified as follows: (i) by deleting the constant region; (ii) by
replacing the constant region with another constant region, e.g., a
constant region meant to increase half-life, stability or affinity
of the antibody, or a constant region from another species or
antibody class; or (iii) by modifying one or more amino acids in
the constant region to alter, for example, the number of
glycosylation sites, effector cell function, Fc receptor (FcR)
binding, complement fixation, among others.
[0118] Methods for altering an antibody constant region are known
in the art. Antibodies with altered function (e.g., altered
affinity for an effector ligand, such as FcR on a cell, or the C1
component of complement) may be produced by replacing at least one
amino acid residue in the constant portion of the antibody with a
different residue (see, e.g., EP 388,151 A1, U.S. Pat. Nos.
5,624,821 and 5,648,260, all of which are hereby incorporated by
reference in their entireties). Similar types of alterations may
also be applied to murine immunoglobulins and immunoglobulins from
other species. For example, it is possible to alter the affinity of
an Fc region of an antibody (e.g., an IgG, such as a human IgG) for
an FcR (e.g., Fc gamma R1) or for C1q binding by replacing the
specified residue(s) with a residue(s) having an appropriate
functionality on its side chain, or by introducing a charged
functional group, such as glutamate or aspartate, or perhaps an
aromatic nonpolar residue such as phenylalanine, tyrosine,
tryptophan or alanine (see, e.g., U.S. Pat. No. 5,624,821).
[0119] Human antibodies to IL-21 and/or IL-21R may additionally be
produced using transgenic nonhuman animals that are modified so as
to produce fully human antibodies rather than the animal's
endogenous antibodies in response to challenge by an antigen. See,
e.g., PCT publication WO 94/02602. The endogenous genes encoding
the heavy and light immunoglobulin chains in the nonhuman host have
been incapacitated, and active loci encoding human heavy and light
chain immunoglobulins are inserted into the host's genome. The
human genes are incorporated, for example, using yeast artificial
chromosomes containing the requisite human DNA segments. An animal
which provides all the desired modifications is then obtained as
progeny by crossbreeding intermediate transgenic animals containing
fewer than the full complement of the modifications. One embodiment
of such a nonhuman animal is a mouse, and is termed the
XENOMOUSE.TM. as disclosed in PCT publications WO 96/33735 and WO
96/34096. This animal produces B cells that secrete fully human
immunoglobulins. The antibodies can be obtained directly from the
animal after immunization with an immunogen of interest, as, for
example, a preparation of a polyclonal antibody, or alternatively
from immortalized B cells derived from the animal, such as
hybridomas producing monoclonal antibodies. Additionally, the genes
encoding the immunoglobulins with human variable regions can be
recovered and expressed to obtain the antibodies directly, or can
be further modified to obtain analogs of antibodies such as, for
example, single chain Fv molecules.
[0120] Other protein-binding molecules may also be employed to
modulate the activity of IL-21 and/or IL-21R. Such protein-binding
molecules include small modular immunopharmaceutical (SMIP.TM.)
drugs (Trubion Pharmaceuticals, Seattle, Wash.). SMIPs are
single-chain polypeptides composed of a binding domain for a
cognate structure such as an antigen, a counterreceptor or the
like, a hinge-region polypeptide having either one or no cysteine
residues, and immunoglobulin CH2 and CH3 domains (see also
www.trubion.com). SMIPs and their uses and applications are
disclosed in, e.g., U.S. Published Patent Appln. Nos. 2003/0118592,
2003/0133939, 2004/0058445, 2005/0136049, 2005/0175614,
2005/0180970, 2005/0186216, 2005/0202012, 2005/0202023,
2005/0202028, 2005/0202534, and 2005/0238646, and related patent
family members thereof, all of which are hereby incorporated by
reference herein in their entireties.
[0121] As discussed herein, neutralizing or nonneutralizing
antibodies (preferably monoclonal antibodies) binding to IL-21R or
IL-21 or a fusion protein thereof may be useful in the treatment of
immune conditions such as fibrosis and fibrosis-associated
conditions.
[0122] Accordingly, the present invention further provides for
compositions comprising an antibody that specifically reacts with
an IL-21 or IL-21R or a fusion protein thereof.
Screening Assays
[0123] The IL-21R or IL-21 polypeptides or fusion proteins of the
invention may also be used to screen for agents that are capable of
binding to IL-21 or IL-21R, and regulating the level of IL-21
and/or IL-21R, e.g., the level of activity of IL-21 and/or IL-21R,
the level of expression of IL-21 and/or IL-21R (e.g., the level of
IL-21 and/or IL-21R gene products), and/or the level of interaction
between IL-21 and IL-21R. Binding assays using a protein of the
invention, or a binding partner thereof, which may be free or
immobilized to a support, are well known in the art. Purified
cell-based or protein-based (cell-free) screening assays may be
used to identify binding partners and/or ligands (natural or
synthetic, e.g., test compounds) to IL-21R or IL-21 polypeptides or
fusion proteins of the invention. For example, IL-21R may be
immobilized in purified form on a carrier and binding of potential
ligands to IL-21R may be measured.
Methods for Diagnosing, Prognosing, and Monitoring the Progress of
Fibrosis and Fibrosis-Associated Disorders, Related to IL-21
[0124] The present invention provides methods for diagnosing,
prognosing, and monitoring the progress (e.g., monitoring the
course of treatment) of disorders, i.e., fibrosis or
fibrosis-associated conditions or disorders, related to IL-21
and/or IL-21R by, e.g., detecting and/or measuring the level of
IL-21 and/or IL-21R, wherein the phrase "level of IL-21 and/or
IL-21R" and equivalents thereof includes, but is not limited to,
(1) the level of expression of IL-21 and/or IL-21R gene products
(e.g., the level of IL-21 and/or IL-21R protein and/or mRNA in a
cell or sample of interest); (2) the level of activity of IL-21
protein and/or IL-21R protein (e.g., Th2 cytokine expression and/or
function in a cell or sample of interest); and (3) the level of
interaction of IL-21 with IL-21R (e.g., in a cell or sample of
interest). For example, the invention provides methods of
diagnosing, prognosing and monitoring, e.g., by detecting the
upregulation or downregulation of IL-21 and/or IL-21R gene products
and/or activity, and/or by measuring the interaction of IL-21 with
IL-21R, etc. (including but not limited to the use of such methods
in human subjects). IL-21 and/or IL-21R levels may also be measured
in a reference cell or sample of interest to produce or obtain a
reference level of IL-21 and/or IL-21R, or such reference level may
be obtained through other methods, or may be generally known, by
one of skill in the art. These methods may be performed by, e.g.,
utilizing prepackaged diagnostic kits comprising at least one of
the group comprising an IL-21 or IL-21R polynucleotide (or
fragments thereof); an IL-21 or IL-21R polypeptide (or fragments
and/or fusion proteins thereof); an antibody to an IL-21 or IL-21R
polypeptide (or derivatives thereof, or antigen-binding fragments
thereof); or modulators of IL-21 or IL-21R polynucleotides and/or
polypeptides as described herein, which may be conveniently used,
for example, in a clinical setting.
[0125] "Diagnostic" or "diagnosing" means identifying the presence
or absence of a pathologic condition. Diagnostic methods include,
but are not limited to, detecting upregulation of the level of
IL-21 and/or IL-21R by determining a test amount of the gene
products (e.g., RNA, cDNA, or polypeptide, including fragments
thereof) of IL-21 and/or IL-21R, by measuring the activity of IL-21
and/or IL-21R, and/or by measuring the level of interaction of
IL-21 with IL-21R, in a biological sample from a subject (human or
nonhuman mammal), and comparing the test amount with, e.g., a
normal amount or range (e.g., an amount or range from an
individual(s) known not to suffer from disorders related to IL-21,
or from an individual known not to suffer from fibrosis or a
fibrosis-associated condition). Although a particular diagnostic
method may not provide a definitive diagnosis of disorders related
to IL-21, it suffices if the method provides a positive indication
that aids in diagnosis.
[0126] The present invention also provides methods for prognosing
such disorders by detecting, for example, the upregulation of
levels of IL-21 and/or IL-21R, e.g., by detecting upregulation of
IL-21 and/or IL-21R gene products and/or activity, and/or by
measuring the level of interaction of IL-21 with IL-21R, etc.
"Prognostic" or "prognosing" means predicting the probable
development and/or severity of a pathologic condition. Prognostic
methods include determining the test amount of a gene product of
IL-21 and/or IL-21R in a biological sample from a subject, and
comparing the test amount to a prognostic amount or range (i.e., an
amount or range from individuals with varying severities of
disorders, i.e., fibrosis and/or a fibrosis-associated condition,
e.g., related to IL-21) for the level of IL-21 and/or IL-21R.
Various amounts related to the level of IL-21 and/or IL-21R in a
test sample are consistent with certain prognoses for disorders,
e.g., fibrosis or fibrosis-associated conditions or disorders,
related to IL-21 and/or IL-21R. The detection of an amount of IL-21
and/or IL-21R level at a particular prognostic level provides a
prognosis for the subject.
[0127] The present invention also provides methods for monitoring
the progress or course of such disorders related to IL-21 by
detecting, for example, the upregulation of IL-21 and/or IL-21R
levels, e.g., by detecting upregulation of IL-21 and/or IL-21R gene
products, activity, and/or the interaction of IL-21 with IL-21R.
Monitoring methods include determining the test amounts of a gene
product of IL-21 in biological samples taken from a subject at a
first and second time, and comparing the amounts. A change in
amount of an IL-21 and/or IL-21R gene product between the first and
second times indicates a change in the course of an IL-21-related
disorder, with a decrease in amount indicating remission of such
disorders, and an increase in amount indicating progression of such
disorders. Such monitoring assays are also useful for evaluating
the efficacy of a particular therapeutic intervention in patients
being treated for fibrosis or a fibrosis-associated disorder.
Measuring the Level of IL-21 and/or IL-21R
[0128] The level of IL-21 and/or IL-21R (e.g., the level of IL-21
and/or IL-21 gene products, activity, and/or interaction) in
methods of the invention (e.g., methods for screening for and/or
identifying a compound for treating, ameliorating, or preventing
fibrosis or a fibrosis-associated condition, methods of diagnosing,
prognosing, and/or monitoring the progress of fibrosis or a
fibrosis-associated condition, and methods of treating,
ameliorating, or preventing fibrosis or a fibrosis-associated
condition) outlined herein may be measured in a variety of
biological samples, including bodily fluids (e.g., whole blood,
plasma, and urine), cells (e.g., whole cells, cell fractions, and
cell extracts), and other tissues. Biological samples also include
sections of tissue, such as biopsies and frozen sections taken for
histological purposes. Preferred biological samples include blood,
plasma, lymph, tissue biopsies, urine, CSF (cerebrospinal fluid),
synovial fluid, and BAL (bronchoalveolar lavage). It will be
appreciated that analysis of a biological sample need not
necessarily require removal of cells or tissue from the subject.
For example, appropriately labeled agents that bind IL-21 and/or
IL-21R gene products (e.g., antibodies, nucleic acids) may be
administered to a subject and visualized (when bound to the target)
using standard imaging technology (e.g., CAT, NMR (MRI), and
PET).
[0129] In the methods of treating, ameliorating, or preventing
fibrosis or a fibrosis-associated disorder, in the methods for
identifying a compound for treating, ameliorating or preventing
fibrosis or a fibrosis-associated disorder in a subject, and in the
diagnostic, prognostic, and monitoring assays and methods of the
present invention, the level of IL-21 and/or IL-21R is detected and
measured to yield a test amount. The test amount is then compared
with, e.g., a normal amount or range. For example, an amount above
(e.g., a higher level) the normal amount or range is a positive
sign in the diagnosis of disorders related to IL-21.
[0130] Normal amounts or baseline levels of IL-21 and/or IL-21R may
be determined for any particular sample type and population.
Generally, baseline (normal) levels of IL-21 and/or IL-21R are
determined by measuring respective levels of IL-21 and/or IL-21R in
a biological sample type from normal (i.e., healthy) subjects.
Alternatively, normal levels of IL-21 and/or IL-21R may be
determined by measuring the amount in healthy cells or tissues
taken from the same subject from which the diseased (or possibly
diseased) test cells or tissues were taken. The level of IL-21
and/or IL-21R (either the normal amount or the test amount) may be
determined or expressed on a per cell, per total protein, or per
volume basis. To determine the cell amount of a sample, one can
measure the level of a constitutively expressed gene product or
other gene product expressed at known levels in cells of the type
from which the biological sample was taken.
[0131] It will be appreciated that the assay methods of the present
invention do not necessarily require measurement of absolute values
for the level of IL-21 and/or IL-21R because relative values are
sufficient for many applications of these methods. It will also be
appreciated that in addition to the quantity or abundance of IL-21
and/or IL-21R levels, variant or abnormal IL-21 and/or IL-21R
levels or their expression patterns (e.g., mutated transcripts,
truncated polypeptides) may be identified by comparison to normal
levels and expression patterns.
[0132] Whether the level of a particular gene or protein in two
samples is increased (i.e., higher) or reduced (i.e., lower), e.g.,
significantly above or significantly below a given level,
respectively, depends on the gene itself and, inter alia, its
variability in expression, activity, and/or interaction with a
ligand between different individuals or different samples. It is
within the skill in the art to determine whether IL-21 and/or
IL-21R levels are significantly similar or different among samples.
Factors such as genetic variation between individuals, species,
organs, tissues, or cells may be taken into consideration (when and
where necessary) for determining whether the level of IL-21 and/or
IL-21R between two samples is increased or reduced. As a result of
the natural heterogeneity in IL-21 and/or IL-21R levels between
individuals, species, organs, tissues, or cells, phrases such as
"significantly above" or "significantly below" cannot be defined as
a precise percentage or value, but rather can be ascertained by one
skilled in the art upon practicing the invention. Particular
methods of detection and measurement of IL-21 and/or IL-21R gene
products, activity, and interaction are described herein.
Assays for Measuring IL-21 and/or IL-21R Gene Products
[0133] The methods of the present invention involve detecting and
quantifying the level of IL-21 and/or IL-21R, e.g., the level of
the gene products of IL-21 and/or IL-21R, the level of activity of
IL-21 and/or IL-21R, and/or the level of interaction of IL-21 with
IL-21R, in biological samples. IL-21 and IL-21R gene products
include mRNAs and polypeptides, and both can be measured using
methods well known to those skilled in the art.
[0134] For example, mRNA can be directly detected and quantified
using hybridization-based assays, such as Northern hybridization,
in situ hybridization, dot and slot blots, and oligonucleotide
arrays. Hybridization-based assays refer to assays in which a probe
nucleic acid is hybridized to a target nucleic acid. In some
formats, the target, the probe, or both are immobilized. The
immobilized nucleic acid may be DNA, RNA, or another
oligonucleotide or polynucleotide, and may comprise naturally or
nonnaturally occurring nucleotides, nucleotide analogs, or
backbones. Methods of selecting nucleic acid probe sequences for
use in the present invention (e.g., based on the nucleic acid
sequence of IL-21) are well known in the art.
[0135] Alternatively, mRNA may be amplified before detection and
quantitation. Such amplification-based assays are well known in the
art and include polymerase chain reaction (PCR),
reverse-transcription-PCR (RT-PCR), PCR-enzyme-linked immunosorbent
assay (PCR-ELISA), and ligase chain reaction (LCR). Primers and
probes for producing and detecting amplified IL-21 gene products
(e.g., mRNA or cDNA) may be readily designed and produced without
undue experimentation by those of skill in the art based on the
nucleic acid sequences of IL-21 and/or IL-21R. Amplified IL-21
and/or IL-21R gene products may be directly analyzed, for example,
by gel electrophoresis; by hybridization to a probe nucleic acid;
by sequencing; by detection of a fluorescent, phosphorescent, or
radioactive signal; or by any of a variety of well-known methods.
In addition, methods are known to those of skill in the art for
increasing the signal produced by amplification of target nucleic
acid sequences. One of skill in the art will recognize that,
whichever amplification method is used, a variety of quantitative
methods known in the art (e.g., quantitative PCR) may be used if
quantitation of gene products is desired.
[0136] An IL-21 and/or IL-21R polypeptide (or fragments thereof)
may be detected using various well-known immunological assays
employing the respective anti-IL-21 and/or IL-21R antibodies that
may be generated as described herein. Immunological assays refer to
assays that utilize an antibody (e.g., polyclonal, monoclonal,
chimeric, humanized, scFv, and/or fragments thereof) that
specifically binds to, e.g., an IL-21 polypeptide (or a fragment
thereof). Such well-known immunological assays suitable for the
practice of the present invention include ELISA, radioimmunoassay
(RIA), immunoprecipitation, immunofluorescence,
fluorescence-activated cell sorting (FACS), and Western blotting.
The ordinarily skilled artisan will also recognize that an IL-21
polypeptide may also be detected using a labeled IL-21R
polypeptide(s). One of skill in the art will understand that the
aforementioned methods may be applied to disorders related to
IL-21, especially fibrosis or a fibrosis-associated condition.
Assays for Measuring the Activity of IL-21 and/or IL-21R
[0137] The activity of IL-21/IL-21R (e.g., in response to
IL-21/IL-21R antagonists as modulators of cytokine production and
cell proliferation/differentiation) can be tested using any one of
a number of routine factor-dependent cell proliferation assays for
cell lines including, without limitation, 32D, DA2, DA1G, T10, B9,
B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RBS, DA1, 123, T1165, HT2,
CTLL2, TF-1, Mole and CMK.
[0138] Assays for T cell or thymocyte proliferation are described,
for example, in Current Protocols in Immunology, Coligan et al.
(eds.) Greene Pub. Assoc. & Wiley-Interscience, NY, N.Y. (1991)
(Chapter 3, "In Vitro assays for Mouse Lymphocyte Function" and
Chapter 7, "Immunologic studies in Humans"); Takai et al. (1986) J.
Immunol. 137:3494-500; Bertagnolli et al. (1990) J. Immunol.
145:1706-12; Bertagnolli et al. (1991) Cell. Immunol. 133:327-41;
Bertagnolli et al. (1992) J. Immunol. 149:3778-83; Bowman et al.
(1994) J. Immunol. 152:1756-61. Assays for cytokine production
and/or proliferation of spleen cells, lymph node cells or
thymocytes are described, for example, in "Polyclonal T cell
stimulation" Kruisbeek and Shevach in Current Protocols in
Immunology, Vol. 1 Coligan et al. (eds.) pp. 3.12.1-14, John Wiley
and Sons, Toronto (1994); and "Measurement of mouse and human
Interferon gamma" Schreiber, R. D. in Current Protocols in
Immunology, Vol. 1 Coligan et al. (eds.) pp. 6.8.1-8, John Wiley
and Sons, Toronto (1994).
[0139] Assays for proliferation and differentiation of
hematopoietic and lymphopoietic cells are described in, for
example, "Measurement of Human and Murine Interleukin 2 and
Interleukin 4" Bottomly et al. in Current Protocols in Immunology,
Vol. 1 Coligan et al. (eds.) pp. 6.3.1-12, John Wiley and Sons,
Toronto (1991); deVries et al. (1991) J. Exp. Med. 173:1205-11;
Moreau et al. (1988) Nature 336:690-92; Greenberger et al. (1983)
Proc. Natl. Acad. Sci. U.S.A. 80:2931-38; "Measurement of mouse and
human interleukin 6" Nordan, R. in Current Protocols in Immunology,
Vol. 1 Coligan et al. (eds.) pp. 6.6.1-5, John Wiley and Sons,
Toronto (1991); Smith et al. (1986) Proc. Natl. Acad. Sci. U.S.A.
83:1857-61; "Measurement of human Interleukin 11" Bennett et al. in
Current Protocols in Immunology, Vol. 1 Coligan et al. (eds.) p.
6.15.1, John Wiley and Sons, Toronto (1991); "Measurement of mouse
and human Interleukin 9" Ciarletta et al. in Current Protocols in
Immunology, Vol. 1. Coligan et al. (eds.) p. 6.13.1, John Wiley and
Sons, Toronto (1991).
[0140] Assays for T cell clone responses to antigens (which will
identify, among others, proteins that affect APC-T cell
interactions as well as direct T cell effects by measuring
proliferation and cytokine production) include, for example, those
described in: Current Protocols in Immunology, Coligan et al.
(eds.) Greene Pub. Assoc. and Wiley-Interscience, NY, N.Y. (1991)
(Chapter 3, "In Vitro assays for Mouse Lymphocyte Function";
Chapter 6, "Cytokines and their cellular receptors"; Chapter 7,
"Immunologic studies in Humans"); Weinberger et al. (1980) Proc.
Natl. Acad. Sci. U.S.A. 77:6091-95; Weinberger et al. (1981) Eur.
J. Immunol. 11:405-11; Takai et al. (1986) J. Immunol.
137:3494-500; Takai et al. (1988) J. Immunol. 140:508-12.
Assays for Measuring the Interaction of IL-21 with IL-21R
[0141] Methods for detecting and/or measuring the level of
interaction of IL-21 with IL-21R are well known in the art. For
example, such interactions between a cytokine and its receptor may
be detected and/or measured with, but not limited to, such
techniques as ELISA, Western blotting, immunoprecipitation, Biacore
analysis, etc.
Pharmaceutical Compositions
[0142] In one aspect, the invention features a method of treating,
ameliorating, or preventing an IL-21-related disorder, i.e.,
fibrosis or a fibrosis-associated condition. The method may
comprise contacting a population of cells with (e.g., by
administering to a subject suffering from or at risk for fibrosis
or a fibrosis-associated disorder) an agent that reduces the level
of IL 21 and/or IL 21R activity in the subject, e.g., an
IL-21/IL-21R antagonist (e.g., an anti-IL-21R antibody, an
anti-IL-21 antibody, an antigen-binding fragment of an anti-IL-21R
antibody, an antigen-binding fragment of an anti-IL-21 antibody,
and a soluble fragment of an IL-21R (e.g., an amino acid sequence
that is at least 90% identical to an amino acid sequence selected
from the group consisting of amino acids 1-538 of SEQ ID NO:2,
amino acids 20-538 of SEQ ID NO:2, amino acids 1-235 of SEQ ID
NO:2, amino acids 20-235 of SEQ ID NO:2, amino acids 1-236 of SEQ
ID NO:2, amino acids 20-236 of SEQ ID NO:2, amino acids 1-529 of
SEQ ID NO:5, amino acids 20-529 of SEQ ID NO:5, amino acids 1-236
of SEQ ID NO:5, and amino acid 20-236 of SEQ ID NO:5)) in an amount
sufficient to inhibit the activity of IL-21 in the cell or
population.
[0143] IL-21/IL-21R antagonists for treating fibrosis or a
fibrosis-associated condition may be used as a pharmaceutical
composition when combined with a pharmaceutically acceptable
carrier. Such a composition may contain, in addition to the
IL-21/IL-21R-antagonists and carrier, various diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well
known in the art. The term "pharmaceutically acceptable" means a
nontoxic material that does not interfere with the effectiveness of
the biological activity of the active ingredient(s). The
characteristics of the carrier will depend on the route of
administration, and are generally well known in the art.
[0144] The pharmaceutical composition of the invention may be in
the form of a liposome in which an IL-21/IL-21R-antagonist(s) is
combined with, in addition to other pharmaceutically acceptable
carriers, amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals,
or lamellar layers which exist in aqueous solution. Suitable lipids
for liposomal formulation include, without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin,
phospholipids, saponin, bile acids, and the like. Preparation of
such liposomal formulations is within the level of skill in the
art, as disclosed, e.g., in U.S. Pat. Nos. 4,235,871, 4,501,728,
4,837,028, and 4,737,323, all of which are incorporated herein by
reference in their entireties.
[0145] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that is sufficient to show a
meaningful subject benefit, e.g., amelioration or reduction of
symptoms of, prevention of, healing of, or increase in rate of
healing of such conditions. When applied to an individual active
ingredient, administered alone, the term refers to that ingredient
alone. When applied to a combination, the term refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or
simultaneously.
[0146] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of an IL-21/IL-21R
antagonist is administered to a subject, e.g., a mammal (e.g., a
human). An IL-21/IL-21R antagonist(s) may be administered in
accordance with the method of the invention either alone or in
combination with other therapies as described in more detail
herein. When coadministered with one or more agents, an IL-21
and/or IL-21R antagonist may be administered either simultaneously
with the second agent, or sequentially. If administered
sequentially, the attending physician will decide on the
appropriate sequence of administering an IL-21/IL-21R antagonist in
combination with other agents.
[0147] Administration of an IL-21/IL-21R antagonist used in a
pharmaceutical composition of the present invention or to practice
a method of the present invention may be carried out in a variety
of conventional ways, such as oral ingestion, inhalation, or
cutaneous, subcutaneous, or intravenous injection. Intravenous
administration to the subject is sometimes preferred. When a
therapeutically effective amount of an IL-21/IL-21R agonist or
antagonist is administered orally, the binding agent will be in the
form of a tablet, capsule, powder, solution or elixir. When
administered in tablet form, the pharmaceutical composition of the
invention may additionally contain a solid carrier such as a
gelatin or an adjuvant. The tablet, capsule, and powder contain
from about 5 to 95% binding agent, and preferably from about 25 to
90% binding agent. When administered in liquid form, a liquid
carrier such as water, petroleum, oils of animal or plant origin
such as peanut oil (albeit keeping in mind the frequency of peanut
allergies in the population), mineral oil, soybean oil, or sesame
oil, or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition
contains from about 0.5 to 90% by weight of the binding agent, and
preferably from about 1 to 50% of the binding agent.
[0148] When a therapeutically effective amount of an IL-21/IL-21R
antagonist is administered by intravenous, cutaneous or
subcutaneous injection, the binding agent will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable protein solutions,
having due regard to pH, isotonicity, stability, and the like, is
within the skill in the art. A preferred pharmaceutical composition
for intravenous, cutaneous, or subcutaneous injection should
contain, in addition to a binding agent, an isotonic vehicle such
as sodium chloride injection, Ringer's injection, dextrose
injection, dextrose and sodium chloride injection, lactated
Ringer's injection, or other vehicle as known in the art. The
pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or other
additive known to those of skill in the art.
[0149] The amount of an IL-21/IL-21R antagonist in the
pharmaceutical composition of the present invention will depend
upon the nature and severity of the condition being treated, and on
the nature of prior treatments that the subject has undergone.
Ultimately, the attending physician will decide the amount of
binding agent with which to treat each individual subject.
Initially, the attending physician will administer low doses of
binding agent and observe the subject's response. Larger doses of
binding agent may be administered until the optimal therapeutic
effect is obtained for the subject, and at that point the dosage is
not generally increased further. It is contemplated that the
various pharmaceutical compositions used to practice the method of
the present invention should contain about 0.1 .mu.g to about 100
mg IL-21/IL-21R antagonist per kg body weight.
[0150] The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the
severity of the disease being treated and the condition and
potential idiosyncratic response of each individual subject. It is
contemplated that the duration of each application of an
IL-21/IL-21R antagonist may be in the range of 12 to 24 hours of
continuous i.v. administration. Also contemplated is subcutaneous
(s.c.) therapy using a pharmaceutical composition of the present
invention. These therapies can be administered daily, weekly, or,
more preferably, biweekly, or monthly. It is also contemplated that
where the IL-21/IL-21R antagonist is a small molecule (e.g., for
oral delivery), the therapies may be administered daily, twice a
day, three times a day, etc. Ultimately the attending physician
will decide on the appropriate duration of i.v. or s.c. therapy, or
therapy with a small molecule, and the timing of administration of
the therapy using the pharmaceutical composition of the present
invention.
[0151] The polynucleotide and proteins of the present invention are
expected to exhibit one or more of the uses or biological
activities (including those associated with assays cited herein)
identified below. Uses or activities described for proteins,
antibodies, or polynucleotides of the present invention may be
provided by administration or use of such proteins, or antibodies,
or by administration or use of polynucleotides encoding such
proteins or antibodies (such as, for example, in gene therapies or
vectors suitable for introduction of DNA).
Combination Therapy
[0152] In one embodiment, a pharmaceutical composition comprising
at least one IL-21R/IL-21 antagonist, e.g., an IL-21R/IL-21
antibody, and at least one therapeutic agent is administered in
combination therapy. Such therapy is useful for treating
pathological conditions or disorders, such as immune and/or
inflammatory disorders. The term "in combination" in this context
means that the antagonist composition and the therapeutic agent are
given substantially contemporaneously, either simultaneously or
sequentially. If given sequentially, at the onset of administration
of the second compound, the first of the two compounds may still be
detectable at effective concentrations at the site of
treatment.
[0153] For example, the combination therapy can include at least
one IL-21R/IL-21 antagonist coformulated with, and/or
coadministered with, at least one additional therapeutic agent.
Additional agents may include at least one cytokine inhibitor,
growth factor inhibitor, immunosuppressant, anti-inflammatory
agent, metabolic inhibitor, enzyme inhibitor, cytotoxic agent, or
cytostatic agent, as described in more detail below. Such
combination therapies may advantageously utilize lower dosages of
the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various
monotherapies. Moreover, the therapeutic agents disclosed herein
act on pathways that differ from the IL-21/IL-21R pathway, and thus
are expected to enhance and/or synergize with the effects of the
IL-21R/IL-21 antagonists.
[0154] Therapeutic agents used in combination with IL-21R/IL-21
antagonists may be those agents that interfere at different stages
in the autoimmune and subsequent inflammatory response. In one
embodiment, at least one IL-21R/IL-21 antagonist described herein
may be coformulated with, and/or coadministered with, at least one
cytokine and/or growth factor antagonist. The cytokine and/or
growth factor antagonists may include soluble receptors, peptide
inhibitors, small molecules, ligand fusions, antibodies (that bind
cytokines or growth factors or their receptors or other cell
surface molecules), and "anti-inflammatory cytokines" and agonists
thereof.
[0155] Nonlimiting examples of the agents that can be used in
combination with the IL-21R/IL-21 antagonists described herein,
include, but are not limited to, antagonists of at least one
interleukin (e.g., IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13,
IL-15, IL-16, IL-17, IL-18, and IL-22); cytokine (e.g., TNF.alpha.,
LT, EMAP-II, and GM-CSF); or growth factor (e.g., FGF and PDGF).
The agents may also include, but are not limited to, antagonists of
at least one receptor for an interleukin, cytokine, and growth
factor. IL-21R/IL-21 antagonists can also be combined with
inhibitors of, e.g., antibodies to, cell surface molecules such as
CD2, CD3, CD4, CD8, CD20 (e.g., the CD20 inhibitor rituximab
(RITUXAN.RTM.)), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1),
CD86 (B7.2), CD90, or their ligands, including CD154 (gp39 or
CD40L), or LFA-1/ICAM-1 and VLA-4/VCAM-1 (Yusuf-Makagiansar et al.
(2002) Med. Res. Rev. 22:146-67). Other compounds that can be used
in combination with IL-21R/IL-21 antagonists described herein may
include antagonists of the receptors for IL-1, IL-12, TNF.alpha.,
IL-15, IL-17, IL-18 and IL-22.
[0156] Examples of agents useful in combination therapies with an
IL-21R/IL-21 antagonist include IL-12 antagonists (such as
antibodies that bind IL-12 (see e.g., WO 00/56772); IL-12 receptor
inhibitors (such as antibodies to the IL-12 receptor); and soluble
IL-12 receptor and fragments thereof. Examples of IL-15 antagonists
include antibodies against IL-15 or its receptor, soluble fragments
of the IL-15 receptor, and IL-15-binding proteins. Examples of
IL-18 antagonists include antibodies to IL-18, soluble fragments of
the IL-18 receptor, and IL-18 binding proteins (IL-18BP, Mallat et
al. (2001) Circ. Res. 89:E41-45). Examples of IL-1 antagonists
include interleukin-1-converting enzyme (ICE) inhibitors (such as
Vx740), IL-1 antagonists (e.g., IL-1RA (anakinra (KINERETT.TM.),
Amgen)), sIL-1RII (Immunex), and anti-IL-1 receptor antibodies.
[0157] Examples of TNF antagonists include antibodies to TNF (e.g.,
human TNF.alpha.), such as D2E7 (human anti-TNF.alpha. antibody,
U.S. Pat. No. 6,258,562, HUMIRA.TM., Abbott Labs);
CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNF.alpha. antibodies,
Celltech/Pharmacia); cA2 (chimeric anti-TNF.alpha. antibody,
REMICADE.TM., Centocor); and anti-TNF antibody fragments (e.g.,
CPD870). Other examples include soluble TNF receptor (e.g., human
p55 or p75) fragments and derivatives, such as p55 kdTNFR-IgG (55
kD TNF receptor-IgG fusion protein, LENERCEPT.TM.) and 75
kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL.TM.
(etanercept--Immunex)). See, e.g., van der Poll et al. (1997)
Blood. 89:3727-34; Mori et al. (1996) J. Immunol. 157:3178-82.
Further examples include enzyme antagonists (e.g., TNF.alpha.
converting enzyme inhibitors (TACE) such as alpha-sulfonyl
hydroxamic acid derivative (WO 01/55112) or N-hydroxyformamide
inhibitors (GW 3333, -005, or -022, GlaxoSmithKline) and
TNF-bp/s-TNFR (soluble TNF binding protein, see, e.g., Lantz et al.
(1991) J Clin Invest. 88:2026-31; Kapadia et al. (1995) Amer. J.
Physiol. Heart Circ. Phys. 268:H517-25). TNF antagonists may be
soluble TNF receptor (e.g., human p55 or p75) fragments and
derivatives, such as 75 kdTNFR-IgG; and TNF.alpha. converting
enzyme (TACE) inhibitors.
[0158] In other embodiments, the IL-21R/IL-21 antagonists described
herein can be administered in combination with at least one of the
following: IL-13 antagonists, such as soluble IL-13 receptors
and/or anti-IL-13 antibodies; and IL-2 antagonists, such as IL-2
fusion proteins (e.g., DAB 486-IL-2 and/or DAB 389-IL-2 made by
Seragen, see e.g., Sewell et al. (1993) Arthritis Rheum.
36:1223-33) and anti-IL-2R antibodies (e.g., anti-Tac (humanized
antibody, Protein Design Labs, see Junghans et al. (1990) Cancer
Res. 50:1495-502). Another combination includes IL-21R/IL-21
antagonists in combination with nondepleting anti-CD4 inhibitors
such as IDEC-CE9.1/SB 210396 (anti-CD4 antibody, GlaxoSmithKline).
Yet other combinations include IL-21R/IL-21 antagonists with CD80
(B7.1) and CD86 (B7.2) costimulatory pathway antagonists (such as
antibodies, soluble receptors, or antagonistic ligands); P-selectin
glycoprotein ligand (PSGL) and PSGL-1 inhibitors (such as
antibodies to PSGL and/or PSGL-1 and small molecule inhibitors); T
cell- and B cell-depleting agents (such as anti-CD4 or anti-CD22
antibodies), and anti-inflammatory cytokines and agonists thereof
(e.g., antibodies). The anti-inflammatory cytokines may include
IL-4 (e.g., Schering-Plough Biopharma); IL-10 (e.g., SCH 52000,
recombinant IL-10, Schering-Plough Biopharma); IL-11; IL-13; and
TGF.beta. or agonists thereof (e.g., agonist antibodies).
[0159] In other embodiments, at least one IL-21R/IL-21 antagonist
can be coformulated with, and/or coadministered with, at least one
anti-inflammatory drug, immunosuppressant, metabolic inhibitor, and
enzymatic inhibitor. Nonlimiting examples of the drugs or
inhibitors that can be used in combination with the IL-21R/IL-21
antagonists described herein, include, but are not limited to, at
least one of: nonsteroidal anti-inflammatory drugs (NSAIDS)
(including, but not limited to, aspirin, salsalate, diflunisal,
ibuprofen, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac,
indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin,
tenidap, meloxicam, piroxicam, aceclofenac, tolmetin, tiaprofenic
acid, nimesulide, etc.); sulfasalazine; corticosteroids (such as
prednisolone); cytokine suppressive anti-inflammatory drugs
(CSAID); inhibitors of nucleotide biosynthesis (such as inhibitors
of purine biosynthesis (e.g., folate antagonist such as
methotrexate)); and inhibitors of pyrimidine biosynthesis, e.g., a
dihydroorotate dehydrogenase (DHODH) inhibitor such as leflunomide
(see, e.g., Kraan et al. (2004) Ann. Rheum. Dis. 63:1056-61).
Therapeutic agents for use in combination with IL-21/IL-21R
antagonists may include one or more NSAIDs, CSAIDs, DHODH
inhibitors (such as leflunomide), and folate antagonists (such as
methotrexate).
[0160] Examples of additional agents that may be used in
combination with IL-21/IL-21R antagonists include at least one of:
corticosteroid (oral, inhaled and local injection);
immunosuppressant (such as cyclosporin and tacrolimus (FK-506)); an
mTOR inhibitor (such as sirolimus (rapamycin) or a rapamycin analog
and/or derivative, e.g., ester rapamycin derivative such as CCI-779
(see, e.g., Elit (2002) Curr. Opin. Investig. Drugs 3:1249-53;
Huang et al. (2002) Curr. Opin. Investig. Drugs 3:295-304)); an
agent which interferes with the signaling of proinflammatory
cytokines such as TNF.alpha. and IL-1 (e.g., an IRAK, NIK, IKK, p38
or MAP kinase inhibitor); TPL-2, Mk-2 and NFKb inhibitors; COX-2
inhibitors (e.g., celecoxib, rofecoxib, etc., and variants
thereof); phosphodiesterase inhibitors (such as Rolipram);
phospholipase inhibitors (e.g., an inhibitor of cytosolic
phospholipase 2 (cPLA2) such as trifluoromethyl ketone analogs
(U.S. Pat. No. 6,350,892)); inhibitors of vascular endothelial cell
growth factor (VEGF); inhibitors of the VEGF receptor; inhibitors
of angiogenesis; RAGE and soluble RAGE; estrogen receptor beta
(ERB) agonists, ERB-NF.kappa.b antagonists; interferon-.beta. (for
example, IFN.beta.-1a and IFN.beta.-1b); copaxone; and
corticosteroids.
[0161] Other useful therapeutic agents that may be combined with an
IL-21R/IL-21 antagonist include: budenoside; epidermal growth
factor; aminosalicylates; 6-mercaptopurine; azathioprine;
metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;
balsalazide; antioxidants; thromboxane inhibitors; growth factors;
elastase inhibitors; pyridinyl-imidazole compounds; glucuronide- or
dextran-conjugated prodrugs of prednisolone; dexamethasone or
budesonide; ICAM-1 antisense phosphorothioate oligodeoxynucleotides
(ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement
receptor 1 (TP10; T Cell Sciences, Inc.); slow-release mesalazine;
antagonists of platelet activating factor (PAF); ciprofloxacin;
lignocaine; cyclosporin A; hydroxychloroquine (PLAQUENIL.TM.);
minocycline (MINOCIN.TM.); and anakinra (KINERETT.TM.).
[0162] Choosing a particular therapeutic agent for administration
in combination with an IL-21/IL-21R antagonist of the invention
will largely depend on factors such as the particular subject, the
desired target, and chosen length of treatment. Such decisions are
well within the skill and knowledge of one skilled in the art.
[0163] Additional examples of therapeutic agents that can be
combined with an IL-21R/IL-21 antagonist include one or more of:
6-mercaptopurines (6-MP); azathioprine; sulphasalazine; mesalazine;
olsalazine; chloroquine, hydroxychloroquine (PLAQUENIL.RTM.);
pencillamine; aurothiornalate (intramuscular and oral);
azathioprine; colchicine; beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeterol); xanthines (theophylline,
aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium
and oxitropium; mycophenolate mofetil; adenosine agonists;
antithrombotic agents; complement inhibitors; and adrenergic
agents.
[0164] In one embodiment, an IL-21R/IL-21 antagonist can be used in
combination with one or more antibodies directed at other targets
involved in regulating immune responses. Nonlimiting examples of
agents for treating or preventing immune responses with which an
IL-21R/IL-21 antagonist of the invention can be combined include
the following: antibodies against other cell surface molecules,
including but not limited to CD25 (interleukin-2 receptor-a), CD11a
(LFA-1), CD54 (ICAM-1), CD4, CD45, CD28, CTLA4, ICOSL, ICOS, CD80
(B7.1), and/or CD86 (B7.2). In yet another embodiment, an
IL-21R/IL-21 antagonist is used in combination with one or more
general immunosuppressive agents, such as cyclosporine A or FK506.
In another embodiment, an IL-21/IL-21R antagonist is used in
combination with a CTLA4 agonist, e.g., (e.g., CTLA4 Ig--abatacept
(ORENCIA.RTM.)).
[0165] The entire contents of all references, patents, and
published patent applications cited throughout this application are
hereby incorporated by reference herein.
EXAMPLES
[0166] The following Examples provide illustrative embodiments of
the invention and do not in any way limit the invention. One of
ordinary skill in the art will recognize that numerous other
embodiments are encompassed within the scope of the invention.
Example 1
Materials and Methods
Example 1.1
Mice, Parasite Infections and Antigen Preparation
[0167] Female or male C57BL/6, C57BL/6/Ai-IL-10KO/IL-4KO mice and
C57BL/6Ai-IL-10KO/IL-12KO were obtained from Taconic Farms
(Germantown, N.Y.) (Hoffmann et al. (1999) J. Immunol.
163:927-938). Breeding pairs of IL-21R.sup.-/- mice on a C57BL/6
background were obtained from a breeding colony housed at Harvard
School of Public Health (Boston, Mass.) (Kasaian et al. (2002)
Immunity 16:559-69). All mice were housed under specific
pathogen-free conditions at the National Institutes of Health in an
American Association for the Accreditation of Laboratory Animal
Care-approved facility. The NIAID animal care and use committee
approved all experimental procedures. S. mansoni eggs were
extracted from the livers of infected mice (Biomedical Research
Institute, Rockville, Md.) as previously described (Wynn et al.
(1995) Nature 376:594-96). For the induction of synchronous primary
pulmonary granulomas, mice were given 5,000 eggs intravenously
(i.v.). For the induction of secondary granulomas, mice were
sensitized intraperitoneally (i.p.) with 5000 live eggs, and then
challenged with 5,000 live eggs i.v. (Wynn et al. (1994) J. Exp.
Med. 179:551-61). In the infection experiments, mice were infected
percutaneously via the tail with 25-30 cercariae of a Puerto-Rican
Strain of S. mansoni (NMRI) that were obtained from infected
Biomphalaria glabrata snails (Biomedical Research Institute,
Rockville, Md.). Soluble egg antigen (SEA) and soluble worm
antigenic preparations (SWAP) were from purified and homogenized
from S. mansoni eggs and adult parasites as previously described
(Cheever et al. (1994) J. Immunol. 153:753-59). All animals
underwent perfusion at the time of sacrifice so that worm and
tissue egg burdens could be determined, as described elsewhere
(id.). Nippostrongylus brasiliensis larvae (L3) were prepared as
previously described (Katona et al. (1983) J. Immunol. 130:350-56).
Mice were inoculated through s.c. injection of 500 L3. On day seven
post-inoculation, lung tissue and mediastinal lymph nodes were
collected for cytokine analysis.
Example 1.2
Histopathology and Fibrosis
[0168] The sizes of pulmonary and hepatic granulomas were
determined on histological sections that were stained with Wright's
Giemsa stain (Histopath of America, Clinton, Md.). Around 30
granulomas per mouse were included in all analyses. A skilled
pathologist evaluated the percentages of eosinophils, mast cells
and other types of cells in the same sections. The number of
schistosome eggs in the liver and the gut and the collagen content
of the liver, as measured by hydroxyproline levels, were determined
as previously described (Cheever et al., supra). Specifically,
hepatic collagen was measured as hydroxyproline by the technique of
Bergman and Loxley (Bergman and Loxley (1963) Analytical Biochem.
35:1961-65) after hydrolysis of a 200-mg portion of liver in 5 ml
of 6N HCl at 110.degree. C. for 18 h. The increase in hepatic
hydroxyproline was positively related to egg numbers in all
experiments and hepatic collagen is reported as the increase above
normal liver collagen in micromoles per 10,000 eggs; (infected
liver collagen--normal liver collagen)/liver eggs.times.10.sup.-4
or micromoles per worm pair. At late chronic time points, fibrosis
is reported as total liver collagen per liver. The same individual
scored all histological features and had no knowledge of the
experimental design.
Example 1.3
FACS Analysis
[0169] Whole lungs were harvested and placed in RPMI. Tissues were
disrupted by straining through a 70-micron nylon mesh (BD Falcon,
San Diego, Calif.). The single cell suspensions were washed and
RBCs were lysed by incubation with ACK lysis solution for 3 min.
Lung lymphocytes were labeled with PE-Cy5 labeled anti-CD4 along
with Fc Block (both antibodies from BD Pharmingen, San Diego,
Calif.) in FACS buffer for 15 min at 4.degree. C. After washing,
the cells were analyzed on a FACS Calibur using FLOWJO.TM. software
(Treestar, Inc., Ashland, Oreg.).
Example 1.4
IL-21 Blocking Experiments with sIL-21R-Fc
[0170] C57BL/6 (10/group) mice were infected percutaneously via the
tail with 30-35 S. mansoni cercariae. Beginning on week 6
post-infection, mice were treated with either mIL-21R-Fc (Wyeth
Research) or Anti-E. tenella murine IgG2a control antibody (Wyeth
Research). Each mouse received one 200 .mu.g dose via i.p.
injection 3.times./week for a total of 5 weeks. Mice were
sacrificed 12 weeks post-infection and hepatic fibrosis was
measured by hydroxyproline assay.
Example 1.5
Lymphocyte Culture and Cytokine Detection Using the Enzyme-linked
Immunosorbent Assay (ELISA)
[0171] Spleen and mesenteric lymph nodes (infection model) or
lung-associated lymph nodes (pulmonary model) were removed
aseptically and single cell suspensions were prepared as previously
described (Hesse et al. (2000) Am. J. Pathol. 157:945-55). Cultures
were incubated at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2. Cells were stimulated with SEA (20 .mu.g/ml), SWAP (50
.mu.g/ml), concanavalin A (Con A; 1 .mu.g/ml), or medium alone.
Supernatant fluids were harvested at 72 hours and assayed for
cytokine production. IFN-.gamma., IL-5 and IL-10 were measured by
sandwich ELISA using paired antibodies (BD Pharmingen, San Diego,
Calif.) as previously described (id.). Cytokine levels were
calculated with standard curves constructed using recombinant
murine cytokines (BD Pharmingen, San Diego, Calif.). IL-13 levels
were measured using murine IL-13 ELISA kits (R&D Systems,
Minneapolis, Minn.) according to the manufacturer's protocol.
TGF-.beta.1 levels were quantified using mouse TGF-.beta.1
DUOSET.RTM. ELISA development system (R&D Systems, Minneapolis,
Minn.) according to manufacture's protocol. To avoid bovine-derived
TGF-.beta.1 contamination, cells were washed 3.times. in PBS and
cultured in media containing 0.5% mouse serum.
Example 1.6
RNA Isolation and Purification and Real-Time Polymerase Chain
Reaction
[0172] Total RNA was extracted from lung and liver tissue samples
placed individually in 1 ml TRIZOL.TM. reagent (Invitrogen,
Carlsbad, Calif.). The sample was homogenized using a tissue
polytron (Omni International Inc., Marietta, Ga.) and total RNA was
extracted according to the recommendations of the manufacturer and
further purified using RNEASY.TM. Mini Kit from Qiagen (Qiagen
Sciences, Germantown, Md.). Individual sample RNA (1 .mu.g) was
reverse-transcribed using SUPERSCRIPT II.TM. (Invitrogen, Carlsbad,
Calif.) and a mixture of oligo (dT) and random primers. Real-time
polymerase chain reaction (RT-PCR) was performed on an ABI
PRISM.TM. 7900 sequence detection system (Applied Biosystems,
Foster City, Calif.). Relative quantities of mRNA for several genes
was determined using SYBR.TM. Green PCR Master Mix (Applied
Biosystems, Foster City, Calif.) and by the comparative threshold
cycle method as described by Applied Biosystems for the ABI
PRISM.TM. 7700/7900 sequence detection systems (Applied Biosystems,
Foster City, Calif.). In this method, mRNA levels for each sample
were normalized to hypoxanthine guanine phosphoribosyl transferase
mRNA levels and then expressed as a relative increase or decrease
compared with levels in uninfected controls. Primers were designed
using PRIMER EXPRESS.TM. software (Applied Biosystems, Foster City,
Calif.). Primers for IL-13, IL-4, IL-10, HPRT (Hesse et al. (2001)
J. Immunol. 167:6533-44), IL-13R.alpha.2 (Chiaramonte et al. (2003)
J. Exp. Med. 197:687-701), Ym1, FIZZ1 and acidic chitinase (AMCase)
(Sandler et al., supra) were published previously, and include:
TABLE-US-00006 IL-21 (SEQ ID NO: 28) 5' GCCAG ATCGC CTCCT GATTA 3'
(sense); (SEQ ID NO: 29) 5' CATGC TCACA GTGCC CCTTT 3' (antisense);
IL-21R (SEQ ID NO: 30) 5' CTCCC CCCTT GAACG TGACT 3' (sense); (SEQ
ID NO: 31) 5' TTGCC CCTCA GCACG TAGTT 3' (antisense); IFN-.gamma.
(SEQ ID NO: 32) 5' AGAGC CAGAT TATCT CTTTC TACCT CAG 3' (sense);
(SEQ ID NO: 33) 5' CCTTT TTCGC CTTGC TGTTG 3' (antisense).
Example 1.7
Serum Antibody Isotype Analysis and Bone Marrow-Derived
Macrophages
[0173] Total IgE was measured using the BD OPTEIA.TM. mouse IgE
ELISA Set (BD Biosciences Pharmingen, San Diego, Calif.) according
to the manufacturer's protocol. SEA-specific IgG1 and IgG2b
isotype-specific antibody (Ab) titers were evaluated by indirect
ELISA. IMMULON.TM. 4 plates (Thermo Labsystems Inc., Beverly Mass.)
were coated with 10 .mu.g/ml SEA (100 .mu.l/well) diluted in PBS,
and serum samples were analyzed using serial two-fold dilutions.
Biotin-Rabbit Anti-mouse IgG1 (Zymed, San Francisco, Calif.) was
used at a 1:1000 dilution. This was followed by peroxidase-labeled
streptavidin (KPL, Gaithersburg, Md.) substrate enzyme at a 1:1000
dilution. Second-step horseradish peroxidase-conjugated rabbit
anti-mouse IgG2b (Zymed, San Francisco, Calif.) Ab was used at a
1:1000 dilution. The absorbance in the wells was read at 405 nm
using a VMAX.TM. Kinetic Microplate Reader (Molecular Devices)
after adding 100 .mu.l one-component ABTS Peroxidase Substrate
(KPL, Gaithersburg, Md.).
[0174] Bone marrow was recovered from female C57BL/6 mice and
cultured in Petri dishes (100.times.15 mm) containing supplemented
DMEM media (L929-conditioned medium) for a period of 6 days. After
six days, cells were harvested and seeded at a concentration of
0.5.times.10.sup.6 cells/well in 24 well plates containing
supplemented DMEM media (10% FBS, 2 mM L-glutamine, 100 U/mL
penicillin, and 100 .mu.g/mL streptomycin). Cells were stimulated
with IL-4, IL-13, and IL-21 (R&D, Minneapolis, Minn.) for a
period of 20 hours. In some assays, cells were pretreated with
IL-21. Cells were lysed and RNA was purified using the RNA Cleanup
procedure with the RNEASY.TM. kits (Qiagen Sciences, Germantown,
Md.).
Example 1.8
Arginase Activity Assay
[0175] Bone marrow-derived macrophages were plated at
6.times.10.sup.5 per well in 96 well tissue culture plates and
stimulated with combinations of IL-4, IL-13, and IL-21. IL-21 was
added 6 hours prior to IL-4 or IL-13 stimulation. Following
stimulation, cells were washed with PBS and lysed with 0.1% Triton
X-100 containing protease inhibitor (Roche, Nutley, N.J.). Lysates
were transferred into a 96 well PCR plate and incubated with 10 mM
MnCl.sub.2 and 50 mM Tris HCl (pH 7.5) to activate enzyme for 10
min at 55.degree. C. After enzyme activation, 25 .mu.l of lysate
was removed and added to 25 .mu.l 1M arginine (pH 9.7) in a new PCR
plate and incubated for 20 hours at 37.degree. C. 5 .mu.l of each
sample was added in duplicate to a 96 well ELISA plate along with 5
.mu.l of each standard, diluted in the same assay conditions,
starting at 100 mg/dL. The urea determination reagent from
QUANTICHROM.TM. Urea Assay Kit (BioAssay Systems, Hayward, Calif.)
was used according to the manufacture's protocol.
Example 1.9
Statistics
[0176] Hepatic fibrosis (adjusted for egg number) decreases with
increasing intensity of infection (worm pairs). Therefore, these
variables were compared by analysis of covariance, using the
logarithm of total liver eggs as the covariate and the logarithm of
hydroxyproline content per egg. Variables that did not change with
infection intensity were compared by one-way ANOVA or Student's t
test (Cheever et al., supra). Changes in cytokine mRNA expression
and granuloma size were evaluated using ANOVA. Differences were
considered significant when p<0.05*, p<0.01**, or
p<0.001***.
Example 2
Regulation of IL-21 and IL-21R during Type-1- and Type-2-Polarized
Responses
[0177] To investigate the regulation and function of the IL-21
receptor in vivo, several different experimental systems of
T.sub.H2-dependent inflammation were examined, including models of
pulmonary and hepatic inflammation, as well as an experimental
model of nematode infection (Pearce et al., supra; Wynn et al.
(1994), supra). In each case, the immune responses of wild type
(WT) animals were compared with IL-21R-deficient mice (Hoffman et
al., supra; Kasaian et al. (2002), supra).
[0178] In comparison to IL-21 (Wurster et al. (2002), supra; Mehta
et al. (2004) Immunol. Rev. 202:84-95), little is known about the
regulation and function of the IL-21 receptor. To determine whether
IL-21 and its receptor are regulated during a pathological T.sub.H2
response in vivo, the S. mansoni model of granuloma formation was
used. In this model, T.sub.H2 cytokines are known to play a
prominent role in lesion formation (Pearce and MacDonald, supra).
Initial studies were designed to determine whether IL-21 and IL-21R
mRNA expression were linked with polarized T.sub.H2 cytokine
responses in vivo. This was achieved by using mice that develop
highly exaggerated T.sub.H1 (IL-4.sup.-/-/IL-10.sup.-/-) or
T.sub.H2 (IL-12.sup.-/-/IL-10.sup.-/-) cytokine responses following
exposure to S. mansoni eggs. In
IL-4.sup.-/-/IL-10.sup.-/-"T.sub.H1" mice, IFN-.gamma. mRNA
expression increased in the lung 75-fold over baseline by day 4
post-challenge and remained approximately 50-fold above background
through day 14 (FIG. 16A). IL-13 mRNA was not detectable in these
mice at any time point, confirming the establishment of a highly
polarized T.sub.H1 inflammatory response. In contrast, the
IL-12.sup.-/-/IL-10.sup.-/-"T.sub.H2" mice displayed a 200 to
250-fold increase in IL-13 mRNA at all time points post-challenge,
with little to no change in IFN-.gamma.. In contrast to the
T.sub.H1/T.sub.H2 cytokines, which displayed a highly polarized
pattern of expression, IL-21 was not associated with a polarized
phenotype (FIG. 16B). In both groups, IL-21 mRNA levels increased
at least 50-fold over baseline following challenge with schistosome
eggs, although the increase observed in the T.sub.H1-polarized mice
was on average 3- to 4-fold greater than the T.sub.H2 polarized
animals (FIG. 16B; lower panel). IL-21R was also not specifically
associated with a T.sub.H1 or T.sub.H2 immune response. However, in
contrast to IL-21, which was more pronounced in T.sub.H1 skewed
animals, the maximal response for the IL-21R was observed in the
T.sub.H2-polarized mice (FIG. 16B; upper panel).
Example 3
Type-2 Cytokine Production is Reduced in the Lungs of
IL-21R-deficient Mice During a Primary Response to Schistosome
Eggs
[0179] Given the significant elevation in IL-21R expression in mice
challenged with schistosome eggs (FIG. 16), the next series of
experiments examined whether IL-21R signaling was influencing the
development of the T.sub.H2 response. In these experiments, naive
WT and IL-21R.sup.-/- mice were injected intravenously with live
schistosome eggs and the production of T.sub.H2 cytokines and
T.sub.H2-regulated genes were monitored in the lung, spleen, and
draining lymph nodes over the following 14 days. In WT mice, IL-21R
mRNA expression increased rapidly following egg exposure and
remained elevated through day 14 (FIG. 17A). IL-21 showed a similar
profile with peak expression occurring on day 7 and then declining
slightly thereafter. Notably, there was a consistent and highly
significant decrease in IL-21 expression on days 7 and 14 in the
IL-21R.sup.-/- mice, suggesting that IL-21R was positively
influencing the expression of its own ligand. Consistent with
previous observations (Wynn et al. (1993) J. Immunol. 151:1430-40;
Vella and Pearce (1992) J. Immunol. 148:2283-88), expression of the
T.sub.H2-associated cytokines IL-4 and IL-13 rose gradually in the
granulomatous tissues of WT mice, with 5- to 15-fold increases
detectable by day 14. In contrast, there was a marked and
significant decrease in IL-4 and IL-13 mRNA expression in the
IL-21R.sup.-/- tissues. Although little change in IFN-.gamma. and
IL-10 mRNA was detected in WT mice between day 4 and 14
post-challenge, production of IFN-.gamma. and IL-10 also decreased
slightly in the IL-21R.sup.-/- animals. Thus, the reduced T.sub.H2
response observed in the IL-21R.sup.-/- mice was not associated
with increased T.sub.H1 cytokine production. The decrease in
T.sub.H2 cytokines was also specific to the granulomatous tissues,
since significant T.sub.H2 cytokine production was observed in
lymph node and splenocyte cultures following in vitro stimulation
with soluble egg antigen (SEA) or mitogen (FIG. 17B). In fact, SEA
consistently stimulated stronger IL-5, IL-10, and IL-13 responses
in the lymph node cultures prepared from IL-21R.sup.-/- mice.
Nevertheless, consistent with the reduced T.sub.H2 response in the
lung, a more rapid resolution of granuloma formation was observed
in the IL-21R.sup.-/- animals (FIG. 17C). In addition, there was a
marked decrease in several genes associated with Stat6-activation
or "alternatively-activated macrophages" (AAMo) (Nair et al. (2005)
Infect. Immun. 73:385-94; Zhu et al. (2004) Science 304:1678-82;
Chiaramonte et al. (2003), supra; Gordon, S. (2003) Nat. Rev.
Immunol. 3:23-35), providing further evidence of an overall
reduction in the T.sub.H2 effector response in IL-21R.sup.-/- mice
(FIG. 17D).
Example 4
TH2 Response is Reduced in IL-21R.sup.-/- Mice Following N.
brasiliensis Infection
[0180] To determine if the reduced T.sub.H2 effector response was
specific to S. mansoni pulmonary granuloma formation, WT and
IL-21R.sup.-/- mice were infected with the intestinal nematode N.
brasiliensis. Infection is established by inoculating third stage
larvae (L3) under the skin. As the parasites mature, they migrate
from the site of inoculation and enter the lungs via the
circulatory system. Once inside the lungs, the parasites trigger a
vigorous and highly polarized T.sub.H2 response (Urban et al.
(1993) J. Immunol. 151:7086-94), which was confirmed by analyzing
the expression of several T.sub.H2-associated genes in the lung
(FIG. 18A) and lung-associated lymph nodes (FIG. 18B). The lungs
and lymph nodes of WT mice displayed marked increases in IL-4,
IL-13, AMCase, FIZZ1/RELM1.alpha., and Ym1 mRNA expression
following N. brasiliensis infection (FIGS. 18A and 18B). However,
in agreement with the pulmonary granuloma model, significantly
reduced levels of IL-4, IL-13, and AMCase were observed, as well as
slightly reduced levels of Ym1 and FIZZ1 mRNA in the lungs of the
IL-21R.sup.-/- mice (FIG. 18A). The draining lymph nodes displayed
a similar reduction, although the decreases in Ym1 and FIZZ1 were
more significant in the lymph nodes (FIG. 18B). The only other
major difference between the two tissues was the AMCase mRNA
response, which appeared to be restricted to the lung. Together,
these data confirm an important role for the IL-21R in T.sub.H2
response development in vivo. Notably however, despite developing a
markedly attenuated T.sub.H2 response, the N. brasiliensis infected
IL-21R.sup.-/- mice displayed no significant delay in adult worm
expulsion (not shown).
Example 5
Type-2 Cytokine-driven Inflammation is Diminished in the Lungs of
IL-21R.sup.-/- Mice
[0181] The next series of experiments were designed to determine
whether the IL-21R modulates the development of secondary T.sub.H2
responses. For these experiments, WT and IL-21R.sup.-/- mice were
sensitized with S. mansoni eggs and challenged intravenously 2
weeks later. As expected, the sensitized mice developed a robust
granulomatous response that was 4- to 5-times greater (FIG. 19C)
than the primary challenge animals (FIG. 17C). As observed in the
primary model, there was a significant increase in IL-21 and IL-21R
mRNA in the lungs following egg exposure, although the IL-21
response peaked much earlier during the secondary challenge. IL-21R
was only modestly increased when compared with IL-21 although it
remained significantly elevated at both time points, while IL-21
mRNA levels declined after reaching a peak on day 4 (FIG. 19A).
Thus, there was evidence of tighter regulation of the ligand in the
tissues. There was also a remarkable decrease in IL-21 expression
in the IL-21R.sup.-/- mice, confirming a potent feedback mechanism
between the receptor and its ligand. Among the T.sub.H2-associated
cytokines, IL-13 was the most robust response, displaying a 50- to
100-fold increase over baseline in WT mice. However, it was reduced
to 10- to 20-fold above background in the IL-21R.sup.-/- mice,
demonstrating that the IL-21R is required for maximum development
of the secondary T.sub.H2 response. Again, the reduction in
T.sub.H2 cytokine expression in IL-21R.sup.-/- mice was not
accompanied by a significant increase in IFN-.gamma.. In fact,
IFN-.gamma. mRNA expression decreased in the lungs of the
IL-21R.sup.-/- mice. Nevertheless, the knockouts displayed a modest
but consistent increase in IFN-.gamma. production in the lymph
nodes and spleen, suggesting a greater inhibition of the T.sub.H2
cytokines overall (FIG. 19B). Consistent with the primary egg
challenge model, the reduction in T.sub.H2 and T.sub.H1 cytokine
production was more pronounced in the granulomatous tissues (FIG.
19A), although the SEA-induced T.sub.H2 response was also partially
reduced in the spleen (FIG. 19B). The significant reduction in
secondary granulomatous inflammation was consistent with the
development of a weaker T.sub.H2 response in the lung (FIG. 19C).
In addition, there was a marked decrease in FIZZ1, Ym1, and AMCase
expression (FIG. 19D), further confirming a significant impairment
of secondary T.sub.H2 effector responses in the IL-21R.sup.-/-
mice.
Example 6
IgG Antibodies, Granuloma Formation, and Type-2 Cytokines Are
Substantially Reduced in Infected IL-21R-deficient Mice
[0182] Next, to determine if IL-21 signaling is required for the
maintenance of a chronic T.sub.H2-dominated response, animals were
exposed percutaneously to S. mansoni cercariae, and their
pathological reactions and immune responses at both acute and
chronic time points post-infection were analyzed. As observed in
the pulmonary granuloma studies, there was a marked upregulation in
IL-21R and IL-21 mRNA expression in the livers of infected WT mice.
In contrast, IL-21 mRNA was almost undetectable in the
IL-21R.sup.-/- mice even after chronic infection (FIG. 20A). At the
acute stage post-infection, the IL-21R.sup.-/- mice also manifested
a marked reduction in T.sub.H2 cytokine mRNA expression (FIG. 20A).
However, the changes were again restricted to the granulomatous
tissues because the lymph node and splenocyte responses of both
groups were similar following in vitro stimulation with parasite
antigens (FIG. 20B). The only consistent difference noted in the in
vitro assays was a 2- to 3-fold decrease in IL-5 and IL-10
production in the splenocyte cultures. The IL-21R.sup.-/- mice also
developed significantly smaller granulomas at the acute stage
post-infection (FIG. 20C), which was consistent with the reduced
IL-4 and IL-13 mRNA responses in the liver (FIG. 20A). However,
this was not accompanied by any obvious change in the percentage of
eosinophils in the granulomas (FIG. 20C). A more detailed
microscopic analysis of the lesions confirmed that there was no
detectable change in the overall composition of the granulomas
(FIG. 21A). Experiments were also undertaken to determine whether
IL-21R-deficiency was specifically affecting the recruitment of
CD4.sup.+ T cells to the granulomatous tissues. To address this
issue, the pulmonary granuloma model was used in order to
synchronize the recruitment of inflammatory cells. However,
consistent with the microscopic evaluations of liver granulomas
(FIG. 21A), the percentage of CD4.sup.+ T cells in the lungs was
similar in WT and IL-21R.sup.-/- mice both before and after egg
exposure (FIG. 21B). Thus, changes in CD4.sup.+ T cell recruitment
or expansion are unlikely to explain the decreased Th1/Th2 cytokine
responses observed in the tissues. Instead, they appear to result
from a more general reduction in the overall inflammatory response.
Importantly, both groups effectively downmodulated their
granulomatous responses by week 12 post-infection (Pearce and
MacDonald, supra). Consequently, there was no significant
difference in granuloma size at the chronic time point (FIG. 20C).
Minimal impairment in the T.sub.H2 cytokine response was observed
in the chronically infected knockout mice (FIG. 20A). The marked
reduction in FIZZ1 and Ym1 observed at the acute stage had also
diminished in the chronically infected IL-21R.sup.-/- animals (FIG.
20D). Nevertheless, expression of AMCase remained remarkably low on
week 12, suggesting a sustained diminution of at least a subset of
the T.sub.H2-driven responses in chronically infected
IL-21R.sup.-/- mice.
[0183] The IL-21R.sup.-/- mice were also examined for changes in
serum antibody levels (FIG. 22). Consistent with their suppressed
cytokine responses (FIG. 20A), the IL-21R.sup.-/- mice displayed a
marked reduction in parasite specific IgG.sub.1
(T.sub.H2-associated antibody) and IgG.sub.2b (T.sub.H1-associated
antibody) titers, which was maintained at the chronic time point
(FIG. 22B). Interestingly however, this was not accompanied by any
significant change in IgE (FIG. 22C), suggesting a selective
impairment in only a subset of serum antibody isotypes. Exogenous
IL-21 has been shown to inhibit IgE production (Suto et al. (2002)
Blood 100:4565-73), which may explain the slight elevation of IgE
in the chronically infected IL-21R.sup.-/- mice. Importantly, the
overall reduction in type-2 responsiveness in the IL-21R.sup.-/-
mice was not attributed to differences in parasite burden since
similar numbers of eggs and paired adult parasites were found in
the tissues of both groups at all time points (FIG. 22A).
Example 7
IL-21R-Deficiency Slows the Progression of Hepatic Fibrosis
[0184] Because T.sub.H2 cytokines are believed to play a major role
in tissue fibrogenesis (Wynn (2004), supra), the development and
progression of hepatic fibrosis in S. mansoni-infected
IL-21R.sup.-/- mice was next examined. Liver hydroxyproline levels
were assayed at various time points post-infection as a direct
measure of tissue collagen content. As expected, marked hepatic
fibrosis was observed in the infected WT mice (FIG. 22D). In
contrast, the IL-21R.sup.-/- displayed significantly less fibrosis
at both the acute and chronic time points. Notably, by week 29
post-infection the IL-21R.sup.-/- mice exhibited more than a 50%
decrease in total liver collagen content compared to WT mice (FIG.
22E), thus confirming an important and indispensable role for the
IL-21R in the progression of T.sub.H2-dependent fibrosis.
[0185] Experiments were undertaken to examine whether an IL-21
inhibitor could slow the progression of fibrosis in infected WT
mice. For these experiments, groups of C57BL/6 mice were treated
with sIL-21R-Fc or control protein for a total of 5 weeks, starting
on week 6 post-infection, around the time when eggs are first
detected in the liver. Although both groups had similar worm and
tissue egg burdens (data not shown), mice receiving the IL-21
blocker displayed over a 50% reduction in hepatic fibrosis at the
termination of the experiment (FIG. 22F). IL-4 and IL-13 mRNA
expression also decreased in the liver, and granuloma size was
reduced approximately 15% (data not shown). Thus, these data
compliment the experiments performed with IL-21R.sup.-/- mice.
Example 8
IL-21 Signaling Promotes the Development of Alternatively Activated
Macrophages
[0186] Because Arg-1, FIZZ1, and TGF-.beta.1 have been linked with
the development of fibrosis, and the expression of several
T.sub.H2/Stat6-regulated genes were reduced in the diseased tissues
of IL-21R.sup.-/- mice (FIGS. 17-20) (see also, Gordon, supra; Nair
et al., supra), experiments were undertaken to determine whether
Arg-1, FIZZ1, and TGF-.beta.1 were directly modulated in
macrophages following stimulation with IL-21. Arg-1 and FIZZ1 are
also well-known markers of alternatively activated macrophages
(AAMo) (Gordon, supra). For these studies, bone marrow-derived
macrophage cultures (BMMo) were generated and then stimulated with
various combinations of IL-4, IL-13, and IL-21. As expected, IL-4
and IL-13 both increased Arg-1 and FIZZ1 mRNA expression, with an
additive effect observed when the two stimuli were used in
combination (FIG. 23A). Notably however, although IL-21 had no
effect on either gene when used alone, cultures that were
pretreated with IL-21 displayed highly significant increases in
Arg-1 and FIZZ1 mRNA expression when subsequently stimulated with
IL-4 and IL-13 (FIG. 23A). The same combination also significantly
increased the function of arginase in the cells (FIG. 23B). In
contrast, IL-21 had no effect on the levels of total or active
TGF-.beta.1 in the culture supernatants (FIG. 24). Unexpectedly,
IL-21 treatment alone significantly increased the expression
IL-4R.alpha. and IL-13R.alpha.1 (FIG. 23C). In contrast, IL-4 and
IL-13 had no effect when used alone (FIG. 23C) and there was no
additional effect when the three stimuli were used in combination
(not shown).
[0187] Because the IL-13R.alpha.2 can also influence
IL-13-dependent signaling (Chiaramonte et al. (2003), supra;
Mentink-Kane et al. Proc. Natl. Acad. Sci. U.S.A. 101:586-90; Wood
et al. (2003) J. Exp. Med. 197:703-09), experiments were undertaken
to examine whether IL-21 was regulating the production of the
IL-13R.alpha.2. Not surprisingly, because the IL-13R.alpha.2 is
primarily produced by nonhematopoietic cells like fibroblasts and
smooth muscle (Chiaramonte et al. (2003), supra; Jakubzick et al.
(2003) Am. J. Pathol. 162:1475-86; Zheng et al. (2003) J. Allergy
Clin. Immunol. 111:720-28; Morimoto et al. (2006) J. Immunol.
176:342-48), there was no evidence of decoy receptor regulation in
the BMMo cultures (data not shown). However, when the regulation of
the decoy receptor in vivo was examined, IL-21 downregulated
IL-13R.alpha.2 mRNA expression in the lungs of i.v. egg-challenged
mice and significantly decreased the levels of the soluble
IL-13R.alpha.2 in their serum (FIG. 23D). When viewed together,
these data suggest that IL-21 contributes to the development of
alternatively activated macrophages by upregulating the type-2 IL-4
receptor (signaling receptor) in macrophages and by simultaneously
decreasing the levels of the soluble IL-13R.alpha.2 (decoy
receptor) in the serum. Both mechanisms likely contributed to the
increased activation of Arg-1 and FIZZ1 in the
IL-4/IL-13-stimulated macrophages. As such, they provide an
additional mechanistic explanation for the impaired T.sub.H2
responses and T.sub.H2-dependent fibrosis in the helminth-infected
IL-21R.sup.-/- mice.
Example 9
Discussion
[0188] IL-21 was recently characterized as a T.sub.H2 cytokine that
can inhibit the differentiation of naive T.sub.H cells into
IFN-.gamma.-producing T.sub.H1 cells (Wurster et al. (2002),
supra). Because the immune response in schistosomiasis evolves from
an early IFN-.gamma. to a sustained and dominant T.sub.H2 response
(Pearce and MacDonald, supra), the influence of IL-21R signaling on
the development of helminth-induced T.sub.H2 responses was
examined. Infection of WT mice with S. mansoni increased IL-21 and
IL-21R expression in the liver, confirming an association of IL-21
signaling with helminth-induced type-2 immunity. However, in the
lung, schistosome eggs induced significant IL-21 expression during
both T.sub.H1 and T.sub.H2 polarized responses. In fact, IL-21
expression increased most when mice were polarized to a T.sub.H1
response. These data suggested that IL-21 exhibits a less
restricted pattern of expression than that of the other
T.sub.H2-associated cytokines. The receptor for IL-21 also failed
to display a T.sub.H1/T.sub.H2-specific pattern. However, the IL-21
receptor was induced nearly 4-fold more in the lungs of T.sub.H2
versus T.sub.H1 polarized mice, which provided one of the first
indications that IL-21R signaling might be involved in the
regulation of T.sub.H2-mediated inflammation.
[0189] To determine whether type-2 effector responses were
compromised in the absence of the IL-21R, the expression of several
genes that are induced preferentially under T.sub.H2-polarizing
conditions was examined. These genes included AMCase, Ym1, and
FIZZ1, all of which are thought to play important and nonredundant
roles in the regulation of T.sub.H2-mediated inflammation (Zhu et
al., supra; Chiaramonte et al. (2003), supra; Nair et al., supra;
Mentink-Kane et al., supra; Guo et al. (2000) J. Biol. Chem.
275:8032-37). Although some variation was observed during a
primary, secondary, or chronic immune response, in each case the
IL-21R.sup.-/- mice displayed highly significant decreases in these
T.sub.H2-associated genes. Ym1 and AMCase are members of a family
of proteins that share homology with chitinases of lower organisms
(Nair et al., supra). Although their exact function in host immune
reactions remains uncertain, they are thought to play important
roles in eosinophil chemotaxis, tissue remodeling and fibrosis.
Indeed, a recent study showed that AMCase neutralization could
ameliorate allergen-driven inflammation and airway
hyperresponsiveness, thus confirming the participation of mammalian
chitinases in T.sub.H2 immunity (Zhu et al, supra). FIZZ1 is also
associated with tissue fibrogenesis (Mentink-Kane et al., supra;
Liu et al. (2004) J. Immunol. 173:3425-31). Consequently, a major
function of the IL-21R may be to regulate the mechanisms of wound
healing and fibrosis. Therefore, in addition to its participation
in helminth-induced immune responses, the IL-21R may be involved in
the regulation of a variety of T.sub.H2-mediated inflammatory
disorders.
[0190] In schistosomiasis, IL-21R-deficiency had a profound effect
on the progression of the disease. Although infection intensities
were the same in WT and IL-21R.sup.-/- mice, the egg-induced
inflammatory response decreased significantly in the absence of the
IL-21R. There was also a marked reduction in secondary granuloma
formation and a faster resolution of primary granulomas in the
lung. Together, these data illustrate an indispensable role for the
IL-21R in granulomatous inflammation. Previous studies showed that
IL-4 and IL-13 are essential for lesion formation (Pearce and
MacDonald; supra), thus the IL-21R is believed to be either
directly or indirectly affecting the activity of these cytokines.
These studies suggested that IL-21 was not acting alone since
extremely high levels of IL-21 were observed in IL-4/IL-10 double
knockout mice, yet granuloma formation was almost completely
ablated in these T.sub.H2-deficient animals (Hoffmann et al. (2000)
J. Immunol. 164:6406-16; Sandler et al. (2003) J. Immunol.
171:3655-67). Thus, IL-21 appears to collaborate with IL-4 and
IL-13 to induce a maximal response. The data disclosed herein show
there is no detectable change in the cellular composition of the
granulomas in IL-21R.sup.-/- mice and no specific impairment in
CD4.sup.+ T cell recruitment. Together, these findings suggested
that the IL-21R regulates the development of parasite-induced
pathology by modulating the overall intensity of the T.sub.H2
effector response.
[0191] IL-21 is not thought to regulate IL-4-induced T.sub.H2 cell
differentiation directly (Suto et al., supra; Wurster et al.
(2002), supra). Instead, it was hypothesized in a recent paper that
IL-21 might amplify T.sub.H2-driven responses by downregulating the
expansion of IFN-.gamma.-producing T.sub.H1 cells (Wurster et al.
(2002), supra). As such, it is theorized that the IFN-.gamma.
response in schistosome-infected mice might increase in the absence
of the IL-21 receptor. Although a small increase was observed in
lung-associated lymph nodes in vitro, IFN-.gamma. production was
consistently reduced in the granulomatous tissues. Thus, the
studies did not show that endogenous IL-21R played a substantial
role in the inhibition of IFN-.gamma. production during helminth
infection. However, the IL-21R.sup.-/- mice simultaneously
generated weaker T.sub.H1 and T.sub.H2 cytokine responses in the
tissues. The significant reduction in IgG.sub.2b
(T.sub.H1-associated) and IgG.sub.i (T.sub.H2-associated) antibody
titers at all times post-infection supports this conclusion. Th2
cytokines were also decreased at the mRNA level in both the lungs
and lymph nodes following N. brasiliensis infection. Indeed, all of
the direct ex vivo data confirmed a marked reduction in T.sub.H2
cytokine expression and function within the affected tissues.
Nevertheless, there was no consistent reduction in T.sub.H2
cytokine production by isolated lymphocytes following antigen
restimulation, which suggests the IL-21R.sup.-/- mice are capable
of generating significant T.sub.H2 responses, at least in vitro.
Thus, the data disclosed herein suggest the IL-21R is selectively
augmenting T.sub.H2 responses in the tissues. In addition to
promoting the T.sub.H2 response, the IL-21R also increased IL-21
production. Thus, the IL-21R appears to operate in an autocrine
fashion to drive T.sub.H2 cytokine expression and type-2 effector
functions in vivo.
[0192] To further elucidate the mechanisms involved, experiments
were undertaken to determine whether IL-21 was directly modulating
macrophage function, because the in vivo data showed a marked
reduction in several genes that have been associated with the
"alternatively-activated" phenotype (Gordon, supra; Mantovani et
al. (2005) Immunity 23:344-46). Macrophages and fibroblasts
exhibiting an alternatively activated phenotype are major cellular
constituents of schistosome granulomas and functional studies
suggested they are critically involved in the progression of the
disease (Hesse et al. (2001), supra). Indeed, an important study by
Brombacher et al. showed that mice that are completely deficient in
alternatively activated macrophages develop lethal egg-induced
pathology following infection with S. mansoni (Herbert et al.
(2004) Immunity 20:623-35). In addition, because macrophage-derived
TGF-.beta.1 has been implicated in the mechanism of IL-13-mediated
fibrosis (Lee et al. (2001) J. Exp. Med. 194:809-21; Fichtner-Feigl
et al. (2006) Nat. Med. 12:99-106), experiments were undertaken to
determine whether IL-21 was modulating TGF-.beta.1 production in
macrophages. To investigate these issues, Arg-1 and FIZZ1 mRNA,
arginase activity, and TGF-.beta.1 protein responses were measured
in bone marrow-derived macrophages following stimulation with
various combinations of IL-21, IL-4, and IL-13. Arg-1 and FIZZ1 are
IL-4R.alpha./Stat6-dependent genes (Liu et al., supra; Hesse et al.
(2001), supra; Munder et al. (1998) J. Immunol. 160:5347-54);
therefore, they serve as functional markers of alternative
macrophage activation. Importantly, the findings suggested that
when macrophages were exposed to IL-21, they became much more
sensitive to the Arg-1- and FIZZ1-inducing activities of IL-4 and
IL-13. Arginase activity assessed by the production of urea also
increased significantly, confirming IL-21 as an important stimulus
for the development of highly functional alternatively activated
macrophages. In contrast, IL-21 had no effect on the production of
TGF-.beta.1 by macrophages. Thus, the pro-fibrotic cytokine
TGF-.beta.1 does appear to be involved, which is consistent with
previous studies that have investigated the role of TGF-.beta.1 in
schistosomiasis (Kaviratne et al. (2004) J. Immunol. 173:4020-29).
Instead, IL-21 significantly increased IL-4R.alpha. and
IL-13R.alpha.1 expression in BMMos and decreased the production of
the soluble IL-13 decoy receptor in vivo, which likely explains
their heightened sensitivity to IL-4 and IL-13. As such, these data
compliment the in vivo studies with IL-21R.sup.-/- mice and suggest
that an important function of IL-21R signaling is to enhance the
development of AAMo, which have been implicated in the mechanism of
fibrosis (Hesse et al. (2001), supra; Hesse et al. (2000), supra).
Moreover, because AAMo have been shown to amplify CD4.sup.+
T.sub.H2 cell differentiation (Bonecchi et al. (1998) Blood
92:2668-71), these data may also explain the overall reduction in
helminth-induced T.sub.H2 activity in the IL-21R.sup.-/- mice.
[0193] In human schistosomiasis, the development of fibrotic liver
pathology is the principle cause of chronic morbidity and mortality
(Pearce and MacDonald, supra; Wynn et al. (2004) Immunol. Rev.
201:156-67). Because the T.sub.H2 cytokine response is known to
play an important role in collagen deposition (Wynn et al. (2004),
supra), a final series of experiments examined the influence of the
IL-21R on the progression of hepatic fibrosis. Notably, development
of fibrosis decreased significantly in the IL-21R.sup.-/- mice,
with the knockout animals displaying over a 50% reduction in
hepatic fibrosis by week 29 post-infection. Importantly, similar
findings were also generated when infected WT mice were treated
with sIL-21R-Fc. Thus, the IL-21 receptor was revealed as a
potential new target for anti-fibrotic therapy. In conclusion,
these studies illustrate an essential role for the IL-21R in the
progression of T.sub.H2 cytokine-mediated disease. As such, the
IL-21R should be added to list of important receptors that regulate
type-2 immunity and macrophage polarization.
Example 10
Prophetic Treatments
[0194] A nonlimiting set of prophetic treatment examples
follows.
[0195] A subject diagnosed with liver cirrhosis is administered an
IL-21R fusion protein to reduce the accumulation of fibrotic tissue
in the liver. The IL-21R fusion protein includes amino acids 1-235
of SEQ ID NO:2 fused at its C-terminus via a linker (corresponding
to amino acids 236-243 of SEQ ID NO:17) to a human immunoglobulin
G1 (IgG1) Fc-mutated sequence (corresponding to amino acids 244-467
of SEQ ID NO:17).
[0196] A subject diagnosed with an infection with schistosoma is
administered a soluble IL-21R fragment to reduce the accumulation
of fibrotic tissue. The fragment contains amino acids 20-538 of SEQ
ID NO:2.
[0197] Following surgery, a subject is administered an IL-21R
antibody to reduce the accumulation of fibrosis due to surgical
incision during the wound healing process.
[0198] A subject diagnosed with liver cirrhosis is administered an
IL-21 antibody to reduce the accumulation of fibrotic tissue in the
liver.
Sequence CWU 1
1
3512665DNAHomo sapiensCDS(236)..(1849) 1gtcgactgga ggcccagctg
cccgtcatca gagtgacagg tcttatgaca gcctgattgg 60tgactcgggc tgggtgtgga
ttctcacccc aggcctctgc ctgctttctc agaccctcat 120ctgtcacccc
cacgctgaac ccagctgcca cccccagaag cccatcagac tgcccccagc
180acacggaatg gatttctgag aaagaagccg aaacagaagg cccgtgggag tcagc atg
238 Met 1ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg ctg ctc cag
gga ggc 286Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln
Gly Gly 5 10 15tgg ggc tgc ccc gac ctc gtc tgc tac acc gat tac ctc
cag acg gtc 334Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu
Gln Thr Val 20 25 30atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc
acg ctc acc ctt 382Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser
Thr Leu Thr Leu 35 40 45acc tgg caa gac cag tat gaa gag ctg aag gac
gag gcc acc tcc tgc 430Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp
Glu Ala Thr Ser Cys50 55 60 65agc ctc cac agg tcg gcc cac aat gcc
acg cat gcc acc tac acc tgc 478Ser Leu His Arg Ser Ala His Asn Ala
Thr His Ala Thr Tyr Thr Cys 70 75 80cac atg gat gta ttc cac ttc atg
gcc gac gac att ttc agt gtc aac 526His Met Asp Val Phe His Phe Met
Ala Asp Asp Ile Phe Ser Val Asn 85 90 95atc aca gac cag tct ggc aac
tac tcc cag gag tgt ggc agc ttt ctc 574Ile Thr Asp Gln Ser Gly Asn
Tyr Ser Gln Glu Cys Gly Ser Phe Leu 100 105 110ctg gct gag agc atc
aag ccg gct ccc cct ttc aac gtg act gtg acc 622Leu Ala Glu Ser Ile
Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr 115 120 125ttc tca gga
cag tat aat atc tcc tgg cgc tca gat tac gaa gac cct 670Phe Ser Gly
Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro130 135 140
145gcc ttc tac atg ctg aag ggc aag ctt cag tat gag ctg cag tac agg
718Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg
150 155 160aac cgg gga gac ccc tgg gct gtg agt ccg agg aga aag ctg
atc tca 766Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu
Ile Ser 165 170 175gtg gac tca aga agt gtc tcc ctc ctc ccc ctg gag
ttc cgc aaa gac 814Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu
Phe Arg Lys Asp 180 185 190tcg agc tat gag ctg cag gtg cgg gca ggg
ccc atg cct ggc tcc tcc 862Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly
Pro Met Pro Gly Ser Ser 195 200 205tac cag ggg acc tgg agt gaa tgg
agt gac ccg gtc atc ttt cag acc 910Tyr Gln Gly Thr Trp Ser Glu Trp
Ser Asp Pro Val Ile Phe Gln Thr210 215 220 225cag tca gag gag tta
aag gaa ggc tgg aac cct cac ctg ctg ctt ctc 958Gln Ser Glu Glu Leu
Lys Glu Gly Trp Asn Pro His Leu Leu Leu Leu 230 235 240ctc ctg ctt
gtc ata gtc ttc att cct gcc ttc tgg agc ctg aag acc 1006Leu Leu Leu
Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys Thr 245 250 255cat
cca ttg tgg agg cta tgg aag aag ata tgg gcc gtc ccc agc cct 1054His
Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser Pro 260 265
270gag cgg ttc ttc atg ccc ctg tac aag ggc tgc agc gga gac ttc aag
1102Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe Lys
275 280 285aaa tgg gtg ggt gca ccc ttc act ggc tcc agc ctg gag ctg
gga ccc 1150Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu
Gly Pro290 295 300 305tgg agc cca gag gtg ccc tcc acc ctg gag gtg
tac agc tgc cac cca 1198Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val
Tyr Ser Cys His Pro 310 315 320cca cgg agc ccg gcc aag agg ctg cag
ctc acg gag cta caa gaa cca 1246Pro Arg Ser Pro Ala Lys Arg Leu Gln
Leu Thr Glu Leu Gln Glu Pro 325 330 335gca gag ctg gtg gag tct gac
ggt gtg ccc aag ccc agc ttc tgg ccg 1294Ala Glu Leu Val Glu Ser Asp
Gly Val Pro Lys Pro Ser Phe Trp Pro 340 345 350aca gcc cag aac tcg
ggg ggc tca gct tac agt gag gag agg gat cgg 1342Thr Ala Gln Asn Ser
Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp Arg 355 360 365cca tac ggc
ctg gtg tcc att gac aca gtg act gtg cta gat gca gag 1390Pro Tyr Gly
Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala Glu370 375 380
385ggg cca tgc acc tgg ccc tgc agc tgt gag gat gac ggc tac cca gcc
1438Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala
390 395 400ctg gac ctg gat gct ggc ctg gag ccc agc cca ggc cta gag
gac cca 1486Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu
Asp Pro 405 410 415ctc ttg gat gca ggg acc aca gtc ctg tcc tgt ggc
tgt gtc tca gct 1534Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly
Cys Val Ser Ala 420 425 430ggc agc cct ggg cta gga ggg ccc ctg gga
agc ctc ctg gac aga cta 1582Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly
Ser Leu Leu Asp Arg Leu 435 440 445aag cca ccc ctt gca gat ggg gag
gac tgg gct ggg gga ctg ccc tgg 1630Lys Pro Pro Leu Ala Asp Gly Glu
Asp Trp Ala Gly Gly Leu Pro Trp450 455 460 465ggt ggc cgg tca cct
gga ggg gtc tca gag agt gag gcg ggc tca ccc 1678Gly Gly Arg Ser Pro
Gly Gly Val Ser Glu Ser Glu Ala Gly Ser Pro 470 475 480ctg gcc ggc
ctg gat atg gac acg ttt gac agt ggc ttt gtg ggc tct 1726Leu Ala Gly
Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly Ser 485 490 495gac
tgc agc agc cct gtg gag tgt gac ttc acc agc ccc ggg gac gaa 1774Asp
Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp Glu 500 505
510gga ccc ccc cgg agc tac ctc cgc cag tgg gtg gtc att cct ccg cca
1822Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro Pro
515 520 525ctt tcg agc cct gga ccc cag gcc agc taatgaggct
gactggatgt 1869Leu Ser Ser Pro Gly Pro Gln Ala Ser530 535ccagagctgg
ccaggccact gggccctgag ccagagacaa ggtcacctgg gctgtgatgt
1929gaagacacct gcagcctttg gtctcctgga tgggcctttg agcctgatgt
ttacagtgtc 1989tgtgtgtgtg tgtgcatatg tgtgtgtgtg catatgcatg
tgtgtgtgtg tgtgtgtctt 2049aggtgcgcag tggcatgtcc acgtgtgtgt
gtgattgcac gtgcctgtgg gcctgggata 2109atgcccatgg tactccatgc
attcacctgc cctgtgcatg tctggactca cggagctcac 2169ccatgtgcac
aagtgtgcac agtaaacgtg tttgtggtca acagatgaca acagccgtcc
2229tccctcctag ggtcttgtgt tgcaagttgg tccacagcat ctccggggct
ttgtgggatc 2289agggcattgc ctgtgactga ggcggagccc agccctccag
cgtctgcctc caggagctgc 2349aagaagtcca tattgttcct tatcacctgc
caacaggaag cgaaagggga tggagtgagc 2409ccatggtgac ctcgggaatg
gcaatttttt gggcggcccc tggacgaagg tctgaatccc 2469gactctgata
ccttctggct gtgctacctg agccaagtcg cctcccctct ctgggctaga
2529gtttccttat ccagacagtg gggaaggcat gacacacctg ggggaaattg
gcgatgtcac 2589ccgtgtacgg tacgcagccc agagcagacc ctcaataaac
gtcagcttcc ttcaaaaaaa 2649aaaaaaaaaa tctaga 26652538PRTHomo sapiens
2Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5
10 15Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln
Thr 20 25 30Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr
Leu Thr 35 40 45Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu
Ala Thr Ser 50 55 60Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His
Ala Thr Tyr Thr65 70 75 80Cys His Met Asp Val Phe His Phe Met Ala
Asp Asp Ile Phe Ser Val 85 90 95Asn Ile Thr Asp Gln Ser Gly Asn Tyr
Ser Gln Glu Cys Gly Ser Phe 100 105 110Leu Leu Ala Glu Ser Ile Lys
Pro Ala Pro Pro Phe Asn Val Thr Val 115 120 125Thr Phe Ser Gly Gln
Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp 130 135 140Pro Ala Phe
Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155
160Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile
165 170 175Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe
Arg Lys 180 185 190Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro
Met Pro Gly Ser 195 200 205Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser
Asp Pro Val Ile Phe Gln 210 215 220Thr Gln Ser Glu Glu Leu Lys Glu
Gly Trp Asn Pro His Leu Leu Leu225 230 235 240Leu Leu Leu Leu Val
Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys 245 250 255Thr His Pro
Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val Pro Ser 260 265 270Pro
Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys Ser Gly Asp Phe 275 280
285Lys Lys Trp Val Gly Ala Pro Phe Thr Gly Ser Ser Leu Glu Leu Gly
290 295 300Pro Trp Ser Pro Glu Val Pro Ser Thr Leu Glu Val Tyr Ser
Cys His305 310 315 320Pro Pro Arg Ser Pro Ala Lys Arg Leu Gln Leu
Thr Glu Leu Gln Glu 325 330 335Pro Ala Glu Leu Val Glu Ser Asp Gly
Val Pro Lys Pro Ser Phe Trp 340 345 350Pro Thr Ala Gln Asn Ser Gly
Gly Ser Ala Tyr Ser Glu Glu Arg Asp 355 360 365Arg Pro Tyr Gly Leu
Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala 370 375 380Glu Gly Pro
Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro385 390 395
400Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser Pro Gly Leu Glu Asp
405 410 415Pro Leu Leu Asp Ala Gly Thr Thr Val Leu Ser Cys Gly Cys
Val Ser 420 425 430Ala Gly Ser Pro Gly Leu Gly Gly Pro Leu Gly Ser
Leu Leu Asp Arg 435 440 445Leu Lys Pro Pro Leu Ala Asp Gly Glu Asp
Trp Ala Gly Gly Leu Pro 450 455 460Trp Gly Gly Arg Ser Pro Gly Gly
Val Ser Glu Ser Glu Ala Gly Ser465 470 475 480Pro Leu Ala Gly Leu
Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly 485 490 495Ser Asp Cys
Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly Asp 500 505 510Glu
Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val Ile Pro Pro 515 520
525Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 530
53535PRTUnknownChemokine receptor conserved motif 3Trp Ser Xaa Trp
Ser1 542628DNAMus musculusCDS(407)..(1993) 4gtcgacgcgg cggtaccagc
tgtctgccca cttctcctgt ggtgtgcctc acggtcactt 60gcttgtctga ccgcaagtct
gcccatccct ggggcagcca actggcctca gcccgtgccc 120caggcgtgcc
ctgtctctgt ctggctgccc cagccctact gtcttcctct gtgtaggctc
180tgcccagatg cccggctggt cctcagcctc aggactatct cagcagtgac
tcccctgatt 240ctggacttgc acctgactga actcctgccc acctcaaacc
ttcacctccc accaccacca 300ctccgagtcc cgctgtgact cccacgccca
ggagaccacc caagtgcccc agcctaaaga 360atggctttct gagaaagacc
ctgaaggagt aggtctggga cacagc atg ccc cgg 415 Met Pro Arg 1ggc cca
gtg gct gcc tta ctc ctg ctg att ctc cat gga gct tgg agc 463Gly Pro
Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly Ala Trp Ser 5 10 15tgc
ctg gac ctc act tgc tac act gac tac ctc tgg acc atc acc tgt 511Cys
Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr Ile Thr Cys20 25 30
35gtc ctg gag aca cgg agc ccc aac ccc agc ata ctc agt ctc acc tgg
559Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser Leu Thr Trp
40 45 50caa gat gaa tat gag gaa ctt cag gac caa gag acc ttc tgc agc
cta 607Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe Cys Ser
Leu 55 60 65cac agg tct ggc cac aac acc aca cat ata tgg tac acg tgc
cat atg 655His Arg Ser Gly His Asn Thr Thr His Ile Trp Tyr Thr Cys
His Met 70 75 80cgc ttg tct caa ttc ctg tcc gat gaa gtt ttc att gtc
aat gtg acg 703Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val
Asn Val Thr 85 90 95gac cag tct ggc aac aac tcc caa gag tgt ggc agc
ttt gtc ctg gct 751Asp Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser
Phe Val Leu Ala100 105 110 115gag agc atc aaa cca gct ccc ccc ttg
aac gtg act gtg gcc ttc tca 799Glu Ser Ile Lys Pro Ala Pro Pro Leu
Asn Val Thr Val Ala Phe Ser 120 125 130gga cgc tat gat atc tcc tgg
gac tca gct tat gac gaa ccc tcc aac 847Gly Arg Tyr Asp Ile Ser Trp
Asp Ser Ala Tyr Asp Glu Pro Ser Asn 135 140 145tac gtg ctg agg ggc
aag cta caa tat gag ctg cag tat cgg aac ctc 895Tyr Val Leu Arg Gly
Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Leu 150 155 160aga gac ccc
tat gct gtg agg ccg gtg acc aag ctg atc tca gtg gac 943Arg Asp Pro
Tyr Ala Val Arg Pro Val Thr Lys Leu Ile Ser Val Asp 165 170 175tca
aga aac gtc tct ctt ctc cct gaa gag ttc cac aaa gat tct agc 991Ser
Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys Asp Ser Ser180 185
190 195tac cag ctg cag gtg cgg gca gcg cct cag cca ggc act tca ttc
agg 1039Tyr Gln Leu Gln Val Arg Ala Ala Pro Gln Pro Gly Thr Ser Phe
Arg 200 205 210ggg acc tgg agt gag tgg agt gac ccc gtc atc ttt cag
acc cag gct 1087Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln
Thr Gln Ala 215 220 225ggg gag ccc gag gca ggc tgg gac cct cac atg
ctg ctg ctc ctg gct 1135Gly Glu Pro Glu Ala Gly Trp Asp Pro His Met
Leu Leu Leu Leu Ala 230 235 240gtc ttg atc att gtc ctg gtt ttc atg
ggt ctg aag atc cac ctg cct 1183Val Leu Ile Ile Val Leu Val Phe Met
Gly Leu Lys Ile His Leu Pro 245 250 255tgg agg cta tgg aaa aag ata
tgg gca cca gtg ccc acc cct gag agt 1231Trp Arg Leu Trp Lys Lys Ile
Trp Ala Pro Val Pro Thr Pro Glu Ser260 265 270 275ttc ttc cag ccc
ctg tac agg gag cac agc ggg aac ttc aag aaa tgg 1279Phe Phe Gln Pro
Leu Tyr Arg Glu His Ser Gly Asn Phe Lys Lys Trp 280 285 290gtt aat
acc cct ttc acg gcc tcc agc ata gag ttg gtg cca cag agt 1327Val Asn
Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val Pro Gln Ser 295 300
305tcc aca aca aca tca gcc tta cat ctg tca ttg tat cca gcc aag gag
1375Ser Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro Ala Lys Glu
310 315 320aag aag ttc ccg ggg ctg ccg ggt ctg gaa gag caa ctg gag
tgt gat 1423Lys Lys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu Glu
Cys Asp 325 330 335gga atg tct gag cct ggt cac tgg tgc ata atc ccc
ttg gca gct ggc 1471Gly Met Ser Glu Pro Gly His Trp Cys Ile Ile Pro
Leu Ala Ala Gly340 345 350 355caa gcg gtc tca gcc tac agt gag gag
aga gac cgg cca tat ggt ctg 1519Gln Ala Val Ser Ala Tyr Ser Glu Glu
Arg Asp Arg Pro Tyr Gly Leu 360 365 370gtg tcc att gac aca gtg act
gtg gga gat gca gag ggc ctg tgt gtc 1567Val Ser Ile Asp Thr Val Thr
Val Gly Asp Ala Glu Gly Leu Cys Val 375 380 385tgg ccc tgt agc tgt
gag gat gat ggc tat cca gcc atg aac ctg gat 1615Trp Pro Cys Ser Cys
Glu Asp Asp Gly Tyr Pro Ala Met Asn Leu Asp 390 395 400gct ggc cga
gag tct ggc cct aat tca gag gat ctg ctc ttg gtc aca 1663Ala Gly Arg
Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu Leu Val Thr 405 410 415gac
cct gct ttt ctg tct tgc ggc tgt gtc tca ggt agt ggt ctc agg 1711Asp
Pro Ala Phe Leu Ser Cys Gly Cys Val Ser Gly Ser Gly Leu Arg420 425
430 435ctt gga ggc tcc cca ggc agc cta ctg gac agg ttg agg ctg tca
ttt 1759Leu Gly Gly Ser Pro Gly Ser Leu Leu Asp Arg Leu Arg Leu Ser
Phe 440 445 450gca aag gaa ggg gac tgg aca gca gac cca acc tgg aga
act ggg tcc 1807Ala Lys Glu Gly Asp Trp Thr Ala Asp Pro Thr Trp Arg
Thr Gly Ser 455 460 465cca gga ggg ggc tct gag agt gaa gca ggt tcc
ccc cct ggt ctg gac 1855Pro Gly Gly Gly Ser Glu Ser Glu Ala Gly Ser
Pro Pro Gly Leu Asp 470 475
480atg gac aca ttt gac agt ggc ttt gca ggt tca gac tgt ggc agc ccc
1903Met Asp Thr Phe Asp Ser Gly Phe Ala Gly Ser Asp Cys Gly Ser Pro
485 490 495gtg gag act gat gaa gga ccc cct cga agc tat ctc cgc cag
tgg gtg 1951Val Glu Thr Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln
Trp Val500 505 510 515gtc agg acc cct cca cct gtg gac agt gga gcc
cag agc agc 1993Val Arg Thr Pro Pro Pro Val Asp Ser Gly Ala Gln Ser
Ser 520 525tagcatataa taaccagcta tagtgagaag aggcctctga gcctggcatt
tacagtgtga 2053acatgtaggg gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt 2113gtgtgtgtgt cttgggttgt gtgttagcac
atccatgttg ggatttggtc tgttgctatg 2173tattgtaatg ctaaattctc
tacccaaagt tctaggccta cgagtgaatt ctcatgttta 2233caaacttgct
gtgtaaacct tgttccttaa tttaatacca ttggttaaat aaaattggct
2293gcaaccaatt actggaggga ttagaggtag ggggcttttg agttacctgt
ttggagatgg 2353agaaggagag aggagagacc aagaggagaa ggaggaagga
gaggagagga gaggagagga 2413gaggagagga gaggagagga gaggagagga
gaggagaggc tgccgtgagg ggagagggac 2473catgagcctg tggccaggag
aaacagcaag tatctggggt acactggtga ggaggtggcc 2533aggccagcag
ttagaagagt agattagggg tgacctccag tatttgtcaa agccaattaa
2593aataacaaaa aaaaaaaaaa agcggccgct ctaga 26285529PRTMus musculus
5Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly1 5
10 15Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp
Thr 20 25 30Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile
Leu Ser 35 40 45Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln
Glu Thr Phe 50 55 60Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His
Ile Trp Tyr Thr65 70 75 80Cys His Met Arg Leu Ser Gln Phe Leu Ser
Asp Glu Val Phe Ile Val 85 90 95Asn Val Thr Asp Gln Ser Gly Asn Asn
Ser Gln Glu Cys Gly Ser Phe 100 105 110Val Leu Ala Glu Ser Ile Lys
Pro Ala Pro Pro Leu Asn Val Thr Val 115 120 125Ala Phe Ser Gly Arg
Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu 130 135 140Pro Ser Asn
Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155
160Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile
165 170 175Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe
His Lys 180 185 190Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro
Gln Pro Gly Thr 195 200 205Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser
Asp Pro Val Ile Phe Gln 210 215 220Thr Gln Ala Gly Glu Pro Glu Ala
Gly Trp Asp Pro His Met Leu Leu225 230 235 240Leu Leu Ala Val Leu
Ile Ile Val Leu Val Phe Met Gly Leu Lys Ile 245 250 255His Leu Pro
Trp Arg Leu Trp Lys Lys Ile Trp Ala Pro Val Pro Thr 260 265 270Pro
Glu Ser Phe Phe Gln Pro Leu Tyr Arg Glu His Ser Gly Asn Phe 275 280
285Lys Lys Trp Val Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val
290 295 300Pro Gln Ser Ser Thr Thr Thr Ser Ala Leu His Leu Ser Leu
Tyr Pro305 310 315 320Ala Lys Glu Lys Lys Phe Pro Gly Leu Pro Gly
Leu Glu Glu Gln Leu 325 330 335Glu Cys Asp Gly Met Ser Glu Pro Gly
His Trp Cys Ile Ile Pro Leu 340 345 350Ala Ala Gly Gln Ala Val Ser
Ala Tyr Ser Glu Glu Arg Asp Arg Pro 355 360 365Tyr Gly Leu Val Ser
Ile Asp Thr Val Thr Val Gly Asp Ala Glu Gly 370 375 380Leu Cys Val
Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met385 390 395
400Asn Leu Asp Ala Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu
405 410 415Leu Val Thr Asp Pro Ala Phe Leu Ser Cys Gly Cys Val Ser
Gly Ser 420 425 430Gly Leu Arg Leu Gly Gly Ser Pro Gly Ser Leu Leu
Asp Arg Leu Arg 435 440 445Leu Ser Phe Ala Lys Glu Gly Asp Trp Thr
Ala Asp Pro Thr Trp Arg 450 455 460Thr Gly Ser Pro Gly Gly Gly Ser
Glu Ser Glu Ala Gly Ser Pro Pro465 470 475 480Gly Leu Asp Met Asp
Thr Phe Asp Ser Gly Phe Ala Gly Ser Asp Cys 485 490 495Gly Ser Pro
Val Glu Thr Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg 500 505 510Gln
Trp Val Val Arg Thr Pro Pro Pro Val Asp Ser Gly Ala Gln Ser 515 520
525Ser 6224PRTUnknownImmunoglobulin Fc Fragment 6His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Leu Gly Ala Pro Ser1 5 10 15Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55
60Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val65
70 75 80Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 85 90 95Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu
Lys Thr 100 105 110Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 115 120 125Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys 130 135 140Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200
205Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 2207617DNAHomo sapiensCDS(47)..(532) 7gctgaagtga aaacgagacc
aaggtctagc tctactgttg gtactt atg aga tcc 55 Met Arg Ser 1agt cct
ggc aac atg gag agg att gtc atc tgt ctg atg gtc atc ttc 103Ser Pro
Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe 5 10 15ttg
ggg aca ctg gtc cac aaa tca agc tcc caa ggt caa gat cgc cac 151Leu
Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His20 25 30
35atg att aga atg cgt caa ctt ata gat att gtt gat cag ctg aaa aat
199Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn
40 45 50tat gtg aat gac ttg gtc cct gaa ttt ctg cca gct cca gaa gat
gta 247Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
Val 55 60 65gag aca aac tgt gag tgg tca gct ttt tcc tgc ttt cag aag
gcc caa 295Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys
Ala Gln 70 75 80cta aag tca gca aat aca gga aac aat gaa agg ata atc
aat gta tca 343Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile
Asn Val Ser 85 90 95att aaa aag ctg aag agg aaa cca cct tcc aca aat
gca ggg aga aga 391Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn
Ala Gly Arg Arg100 105 110 115cag aaa cac aga cta aca tgc cct tca
tgt gat tct tat gag aaa aaa 439Gln Lys His Arg Leu Thr Cys Pro Ser
Cys Asp Ser Tyr Glu Lys Lys 120 125 130cca ccc aaa gaa ttc cta gaa
aga ttc aaa tca ctt ctc caa aag atg 487Pro Pro Lys Glu Phe Leu Glu
Arg Phe Lys Ser Leu Leu Gln Lys Met 135 140 145att cat cag cat ctg
tcc tct aga aca cac gga agt gaa gat tcc 532Ile His Gln His Leu Ser
Ser Arg Thr His Gly Ser Glu Asp Ser 150 155 160tgaggatcta
acttgcagtt ggacactatg ttacatactc taatatagta gtgaaagtca
592tttctttgta ttccaagtgg aggag 6178162PRTHomo sapiens 8Met Arg Ser
Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met1 5 10 15Val Ile
Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln 20 25 30Asp
Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln 35 40
45Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro
50 55 60Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe
Gln65 70 75 80Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu
Arg Ile Ile 85 90 95Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro
Ser Thr Asn Ala 100 105 110Gly Arg Arg Gln Lys His Arg Leu Thr Cys
Pro Ser Cys Asp Ser Tyr 115 120 125Glu Lys Lys Pro Pro Lys Glu Phe
Leu Glu Arg Phe Lys Ser Leu Leu 130 135 140Gln Lys Met Ile His Gln
His Leu Ser Ser Arg Thr His Gly Ser Glu145 150 155 160Asp
Ser916PRTApis mellifera 9Met Pro Leu Leu Leu Leu Leu Leu Leu Leu
Pro Ser Pro Leu His Pro1 5 10 1510786DNAHomo sapiensCDS(1)..(780)
10atg aaa ttc tta gtc aac gtt gcc ctt gtt ttt atg gtc gtg tac att
48Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile1
5 10 15tct tac atc tat gcc ggc agc gga cac cac cat cat cac cac ggt
agc 96Ser Tyr Ile Tyr Ala Gly Ser Gly His His His His His His Gly
Ser 20 25 30ggc gac tat aaa gac gat gac gat aag ggt tcc gga tgc ccc
gac ctc 144Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gly Cys Pro
Asp Leu 35 40 45gtc tgc tac acc gat tac ctc cag acg gtc atc tgc atc
ctg gaa atg 192Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile
Leu Glu Met 50 55 60tgg aac ctc cac ccc agc acg ctc acc ctt acc tgg
caa gac cag tat 240Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp
Gln Asp Gln Tyr65 70 75 80gaa gag ctg aag gac gag gcc acc tcc tgc
agc ctc cac agg tcg gcc 288Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys
Ser Leu His Arg Ser Ala 85 90 95cac aat gcc acg cat gcc acc tac acc
tgc cac atg gat gta ttc cac 336His Asn Ala Thr His Ala Thr Tyr Thr
Cys His Met Asp Val Phe His 100 105 110ttc atg gcc gac gac att ttc
agt gtc aac atc aca gac cag tct ggc 384Phe Met Ala Asp Asp Ile Phe
Ser Val Asn Ile Thr Asp Gln Ser Gly 115 120 125aac tac tcc cag gag
tgt ggc agc ttt ctc ctg gct gag agc atc aag 432Asn Tyr Ser Gln Glu
Cys Gly Ser Phe Leu Leu Ala Glu Ser Ile Lys 130 135 140ccg gct ccc
cct ttc aac gtg act gtg acc ttc tca gga cag tat aat 480Pro Ala Pro
Pro Phe Asn Val Thr Val Thr Phe Ser Gly Gln Tyr Asn145 150 155
160atc tcc tgg cgc tca gat tac gaa gac cct gcc ttc tac atg ctg aag
528Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe Tyr Met Leu Lys
165 170 175ggc aag ctt cag tat gag ctg cag tac agg aac cgg gga gac
ccc tgg 576Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg Gly Asp
Pro Trp 180 185 190gct gtg agt ccg agg aga aag ctg atc tca gtg gac
tca aga agt gtc 624Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp
Ser Arg Ser Val 195 200 205tcc ctc ctc ccc ctg gag ttc cgc aaa gac
tcg agc tat gag ctg cag 672Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp
Ser Ser Tyr Glu Leu Gln 210 215 220gtg cgg gca ggg ccc atg cct ggc
tcc tcc tac cag ggg acc tgg agt 720Val Arg Ala Gly Pro Met Pro Gly
Ser Ser Tyr Gln Gly Thr Trp Ser225 230 235 240gaa tgg agt gac ccg
gtc atc ttt cag acc cag tca gag gag tta aag 768Glu Trp Ser Asp Pro
Val Ile Phe Gln Thr Gln Ser Glu Glu Leu Lys 245 250 255gaa ggc tgg
aac taatga 786Glu Gly Trp Asn 26011260PRTHomo sapiens 11Met Lys Phe
Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile1 5 10 15Ser Tyr
Ile Tyr Ala Gly Ser Gly His His His His His His Gly Ser 20 25 30Gly
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gly Cys Pro Asp Leu 35 40
45Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile Leu Glu Met
50 55 60Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Gln Asp Gln
Tyr65 70 75 80Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His
Arg Ser Ala 85 90 95His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met
Asp Val Phe His 100 105 110Phe Met Ala Asp Asp Ile Phe Ser Val Asn
Ile Thr Asp Gln Ser Gly 115 120 125Asn Tyr Ser Gln Glu Cys Gly Ser
Phe Leu Leu Ala Glu Ser Ile Lys 130 135 140Pro Ala Pro Pro Phe Asn
Val Thr Val Thr Phe Ser Gly Gln Tyr Asn145 150 155 160Ile Ser Trp
Arg Ser Asp Tyr Glu Asp Pro Ala Phe Tyr Met Leu Lys 165 170 175Gly
Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro Trp 180 185
190Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp Ser Arg Ser Val
195 200 205Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr Glu
Leu Gln 210 215 220Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln
Gly Thr Trp Ser225 230 235 240Glu Trp Ser Asp Pro Val Ile Phe Gln
Thr Gln Ser Glu Glu Leu Lys 245 250 255Glu Gly Trp Asn
260121472DNAHomo sapiensCDS(15)..(1415) 12gcggccgcac cacc atg ccg
cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg 50 Met Pro Arg Gly Trp Ala
Ala Pro Leu Leu Leu Leu 1 5 10ctg ctc cag gga ggc tgg ggc tgc ccc
gac ctc gtc tgc tac acc gat 98Leu Leu Gln Gly Gly Trp Gly Cys Pro
Asp Leu Val Cys Tyr Thr Asp 15 20 25tac ctc cag acg gtc atc tgc atc
ctg gaa atg tgg aac ctc cac ccc 146Tyr Leu Gln Thr Val Ile Cys Ile
Leu Glu Met Trp Asn Leu His Pro 30 35 40agc acg ctc acc ctt acc tgg
caa gac cag tat gaa gag ctg aag gac 194Ser Thr Leu Thr Leu Thr Trp
Gln Asp Gln Tyr Glu Glu Leu Lys Asp45 50 55 60gag gcc acc tcc tgc
agc ctc cac agg tcg gcc cac aat gcc acg cat 242Glu Ala Thr Ser Cys
Ser Leu His Arg Ser Ala His Asn Ala Thr His 65 70 75gcc acc tac acc
tgc cac atg gat gta ttc cac ttc atg gcc gac gac 290Ala Thr Tyr Thr
Cys His Met Asp Val Phe His Phe Met Ala Asp Asp 80 85 90att ttc agt
gtc aac atc aca gac cag tct ggc aac tac tcc cag gag 338Ile Phe Ser
Val Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu 95 100 105tgt
ggc agc ttt ctc ctg gct gag agc atc aag ccg gct ccc cct ttc 386Cys
Gly Ser Phe Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe 110 115
120aac gtg act gtg acc ttc tca gga cag tat aat atc tcc tgg cgc tca
434Asn Val Thr Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser125 130 135 140gat tac gaa gac cct gcc ttc tac atg ctg aag ggc
aag ctt cag tat 482Asp Tyr Glu Asp Pro Ala Phe Tyr Met Leu Lys Gly
Lys Leu Gln Tyr 145 150 155gag ctg cag tac agg aac cgg gga gac ccc
tgg gct gtg agt ccg agg 530Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro
Trp Ala Val Ser Pro Arg 160 165 170aga aag ctg atc tca gtg gac tca
aga agt gtc tcc ctc ctc ccc ctg 578Arg Lys Leu Ile Ser Val Asp Ser
Arg Ser Val Ser Leu Leu Pro Leu 175 180 185gag ttc cgc aaa gac tcg
agc tat gag ctg cag gtg cgg gca ggg ccc 626Glu Phe Arg Lys Asp Ser
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro 190 195 200atg cct ggc tcc
tcc tac cag ggg acc tgg agt
gaa tgg agt gac ccg 674Met Pro Gly Ser Ser Tyr Gln Gly Thr Trp Ser
Glu Trp Ser Asp Pro205 210 215 220gtc atc ttt cag acc cag tca gag
gag tta aag gaa ggc tgg aac ggc 722Val Ile Phe Gln Thr Gln Ser Glu
Glu Leu Lys Glu Gly Trp Asn Gly 225 230 235tcc ggc tct aga gac aaa
act cac aca tgc cca ccg tgc cca gca cct 770Ser Gly Ser Arg Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro 240 245 250gaa ctc ctg ggg
gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 818Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 255 260 265gac acc
ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 866Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 270 275
280gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac
914Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp285 290 295 300ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg
gag gag cag tac 962Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr 305 310 315aac agc acg tac cgt gtg gtc agc gtc ctc
acc gtc ctg cac cag gac 1010Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 320 325 330tgg ctg aat ggc aag gag tac aag
tgc aag gtc tcc aac aaa gcc ctc 1058Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu 335 340 345cca gtc ccc atc gag aaa
acc atc tcc aaa gcc aaa ggg cag ccc cga 1106Pro Val Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 350 355 360gaa cca cag gtg
tac acc ctg ccc cca tcc cgg gag gag atg acc aag 1154Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys365 370 375 380aac
cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 1202Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390
395atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag
1250Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
400 405 410acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc
tat agc 1298Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 415 420 425aag ctc acc gtg gac aag agc agg tgg cag cag ggg
aac gtc ttc tca 1346Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser 430 435 440tgc tcc gtg atg cat gag gct ctg cac aac
cac tac acg cag aag agc 1394Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser445 450 455 460ctc tcc ctg tcc ccg ggt aaa
tgagtgaatt cacacgcaga agagcctctc 1445Leu Ser Leu Ser Pro Gly Lys
465cctgtccccg ggtaaatgag tgaattc 147213467PRTHomo sapiens 13Met Pro
Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15Gly
Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr 20 25
30Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr
35 40 45Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr
Ser 50 55 60Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr
Tyr Thr65 70 75 80Cys His Met Asp Val Phe His Phe Met Ala Asp Asp
Ile Phe Ser Val 85 90 95Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln
Glu Cys Gly Ser Phe 100 105 110Leu Leu Ala Glu Ser Ile Lys Pro Ala
Pro Pro Phe Asn Val Thr Val 115 120 125Thr Phe Ser Gly Gln Tyr Asn
Ile Ser Trp Arg Ser Asp Tyr Glu Asp 130 135 140Pro Ala Phe Tyr Met
Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160Arg Asn
Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile 165 170
175Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys
180 185 190Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro
Gly Ser 195 200 205Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro
Val Ile Phe Gln 210 215 220Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp
Asn Gly Ser Gly Ser Arg225 230 235 240Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295
300His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr305 310 315 320Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Val Pro Ile 340 345 350Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 355 360 365Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu385 390 395 400Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410
415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
420 425 430Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 435 440 445His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 450 455 460Pro Gly Lys465141499DNAHomo
sapiensCDS(15)..(1493) 14gcggccgcac cacc atg ccg cgt ggc tgg gcc
gcc ccc ttg ctc ctg ctg 50 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu
Leu Leu 1 5 10ctg ctc cag gga ggc tgg ggc tgc ccc gac ctc gtc tgc
tac acc gat 98Leu Leu Gln Gly Gly Trp Gly Cys Pro Asp Leu Val Cys
Tyr Thr Asp 15 20 25tac ctc cag acg gtc atc tgc atc ctg gaa atg tgg
aac ctc cac ccc 146Tyr Leu Gln Thr Val Ile Cys Ile Leu Glu Met Trp
Asn Leu His Pro 30 35 40agc acg ctc acc ctt acc tgg caa gac cag tat
gaa gag ctg aag gac 194Ser Thr Leu Thr Leu Thr Trp Gln Asp Gln Tyr
Glu Glu Leu Lys Asp45 50 55 60gag gcc acc tcc tgc agc ctc cac agg
tcg gcc cac aat gcc acg cat 242Glu Ala Thr Ser Cys Ser Leu His Arg
Ser Ala His Asn Ala Thr His 65 70 75gcc acc tac acc tgc cac atg gat
gta ttc cac ttc atg gcc gac gac 290Ala Thr Tyr Thr Cys His Met Asp
Val Phe His Phe Met Ala Asp Asp 80 85 90att ttc agt gtc aac atc aca
gac cag tct ggc aac tac tcc cag gag 338Ile Phe Ser Val Asn Ile Thr
Asp Gln Ser Gly Asn Tyr Ser Gln Glu 95 100 105tgt ggc agc ttt ctc
ctg gct gag agc atc aag ccg gct ccc cct ttc 386Cys Gly Ser Phe Leu
Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe 110 115 120aac gtg act
gtg acc ttc tca gga cag tat aat atc tcc tgg cgc tca 434Asn Val Thr
Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser125 130 135
140gat tac gaa gac cct gcc ttc tac atg ctg aag ggc aag ctt cag tat
482Asp Tyr Glu Asp Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr
145 150 155gag ctg cag tac agg aac cgg gga gac ccc tgg gct gtg agt
ccg agg 530Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro Trp Ala Val Ser
Pro Arg 160 165 170aga aag ctg atc tca gtg gac tca aga agt gtc tcc
ctc ctc ccc ctg 578Arg Lys Leu Ile Ser Val Asp Ser Arg Ser Val Ser
Leu Leu Pro Leu 175 180 185gag ttc cgc aaa gac tcg agc tat gag ctg
cag gtg cgg gca ggg ccc 626Glu Phe Arg Lys Asp Ser Ser Tyr Glu Leu
Gln Val Arg Ala Gly Pro 190 195 200atg cct ggc tcc tcc tac cag ggg
acc tgg agt gaa tgg agt gac ccg 674Met Pro Gly Ser Ser Tyr Gln Gly
Thr Trp Ser Glu Trp Ser Asp Pro205 210 215 220gtc atc ttt cag acc
cag tca gag gag tta aag gaa ggc tgg aac ggc 722Val Ile Phe Gln Thr
Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Gly 225 230 235tcc ggc tct
aga gac aaa act cac aca tgc cca ccg tgc cca gca cct 770Ser Gly Ser
Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 240 245 250gaa
ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 818Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 255 260
265gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg
866Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
270 275 280gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac
gtg gac 914Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp285 290 295 300ggc gtg gag gtg cat aat gcc aag aca aag ccg
cgg gag gag cag tac 962Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 305 310 315aac agc acg tac cgt gtg gtc agc gtc
ctc acc gtc ctg cac cag gac 1010Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 320 325 330tgg ctg aat ggc aag gag tac
aag tgc aag gtc tcc aac aaa gcc ctc 1058Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 335 340 345cca gtc ccc atc gag
aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1106Pro Val Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 350 355 360gaa cca cag
gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag 1154Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys365 370 375
380aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac
1202Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac
tac aag 1250Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 400 405 410acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc
ttc ctc tat agc 1298Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 415 420 425aag ctc acc gtg gac aag agc agg tgg cag
cag ggg aac gtc ttc tca 1346Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser 430 435 440tgc tcc gtg atg cat gag gct ctg
cac aac cac tac acg cag aag agc 1394Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser445 450 455 460ctc tcc ctg tcc ccg
ggt aaa tca gga atg gca tca atg aca gga ggt 1442Leu Ser Leu Ser Pro
Gly Lys Ser Gly Met Ala Ser Met Thr Gly Gly 465 470 475caa caa atg
ggt tct gga tct cat cat cat cat cat cat tct gga ggt 1490Gln Gln Met
Gly Ser Gly Ser His His His His His His Ser Gly Gly 480 485 490tga
gaattc 149915492PRTHomo sapiens 15Met Pro Arg Gly Trp Ala Ala Pro
Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15Gly Trp Gly Cys Pro Asp Leu
Val Cys Tyr Thr Asp Tyr Leu Gln Thr 20 25 30Val Ile Cys Ile Leu Glu
Met Trp Asn Leu His Pro Ser Thr Leu Thr 35 40 45Leu Thr Trp Gln Asp
Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60Cys Ser Leu His
Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75 80Cys His
Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95Asn
Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105
110Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val
115 120 125Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr
Glu Asp 130 135 140Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr
Glu Leu Gln Tyr145 150 155 160Arg Asn Arg Gly Asp Pro Trp Ala Val
Ser Pro Arg Arg Lys Leu Ile 165 170 175Ser Val Asp Ser Arg Ser Val
Ser Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190Asp Ser Ser Tyr Glu
Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205Ser Tyr Gln
Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220Thr
Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Gly Ser Gly Ser Arg225 230
235 240Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly 245 250 255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 275 280 285Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile 340 345
350Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
355 360 365Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 370 375 380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 405 410 415Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460Pro
Gly Lys Ser Gly Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly465 470
475 480Ser Gly Ser His His His His His His Ser Gly Gly 485
490161426DNAHomo sapiensCDS(15)..(1415) 16gcggccgcac cacc atg ccg
cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg 50 Met Pro Arg Gly Trp Ala
Ala Pro Leu Leu Leu Leu 1 5 10ctg ctc cag gga ggc tgg ggc tgc ccc
gac ctc gtc tgc tac acc gat 98Leu Leu Gln Gly Gly Trp Gly Cys Pro
Asp Leu Val Cys Tyr Thr Asp 15 20 25tac ctc cag acg gtc atc tgc atc
ctg gaa atg tgg aac ctc cac ccc 146Tyr Leu Gln Thr Val Ile Cys Ile
Leu Glu Met Trp Asn Leu His Pro 30 35 40agc acg ctc acc ctt acc tgg
caa gac cag tat gaa gag ctg aag gac 194Ser Thr Leu Thr Leu Thr Trp
Gln Asp Gln Tyr Glu Glu Leu Lys Asp45 50 55 60gag gcc acc tcc tgc
agc ctc cac agg tcg gcc cac aat gcc acg cat 242Glu Ala Thr Ser Cys
Ser Leu His Arg Ser Ala His Asn Ala Thr His 65 70 75gcc acc tac acc
tgc cac atg gat gta ttc cac ttc atg gcc gac gac 290Ala Thr Tyr Thr
Cys His Met Asp Val Phe His Phe Met Ala Asp Asp 80 85 90att ttc agt
gtc aac atc aca gac cag tct ggc aac tac tcc cag gag 338Ile Phe Ser
Val Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu 95 100 105tgt
ggc agc ttt ctc ctg gct gag agc atc aag ccg gct ccc cct ttc 386Cys
Gly Ser Phe Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe 110 115
120aac gtg act gtg acc ttc tca gga cag tat aat atc tcc tgg cgc tca
434Asn Val Thr Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser125 130 135 140gat tac gaa gac cct gcc ttc tac atg ctg aag ggc
aag ctt cag tat 482Asp Tyr Glu Asp Pro Ala Phe Tyr Met Leu Lys Gly
Lys Leu Gln Tyr 145 150 155gag ctg cag tac agg aac cgg gga gac ccc
tgg gct gtg agt ccg agg
530Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg
160 165 170aga aag ctg atc tca gtg gac tca aga agt gtc tcc ctc ctc
ccc ctg 578Arg Lys Leu Ile Ser Val Asp Ser Arg Ser Val Ser Leu Leu
Pro Leu 175 180 185gag ttc cgc aaa gac tcg agc tat gag ctg cag gtg
cgg gca ggg ccc 626Glu Phe Arg Lys Asp Ser Ser Tyr Glu Leu Gln Val
Arg Ala Gly Pro 190 195 200atg cct ggc tcc tcc tac cag ggg acc tgg
agt gaa tgg agt gac ccg 674Met Pro Gly Ser Ser Tyr Gln Gly Thr Trp
Ser Glu Trp Ser Asp Pro205 210 215 220gtc atc ttt cag acc cag tca
gag gag tta aag gaa ggc tgg aac ggc 722Val Ile Phe Gln Thr Gln Ser
Glu Glu Leu Lys Glu Gly Trp Asn Gly 225 230 235tcc ggc tct aga gac
aaa act cac aca tgc cca ccg tgc cca gca cct 770Ser Gly Ser Arg Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 240 245 250gaa gcc ctg
ggg gca ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 818Glu Ala Leu
Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 255 260 265gac
acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 866Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 270 275
280gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac
914Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp285 290 295 300ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg
gag gag cag tac 962Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr 305 310 315aac agc acg tac cgt gtg gtc agc gtc ctc
acc gtc ctg cac cag gac 1010Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 320 325 330tgg ctg aat ggc aag gag tac aag
tgc aag gtc tcc aac aaa gcc ctc 1058Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu 335 340 345cca gcc ccc atc gag aaa
acc atc tcc aaa gcc aaa ggg cag ccc cga 1106Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 350 355 360gaa cca cag gtg
tac acc ctg ccc cca tcc cgg gag gag atg acc aag 1154Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys365 370 375 380aac
cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 1202Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390
395atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag
1250Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
400 405 410acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc
tat agc 1298Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 415 420 425aag ctc acc gtg gac aag agc agg tgg cag cag ggg
aac gtc ttc tca 1346Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser 430 435 440tgc tcc gtg atg cat gag gct ctg cac aac
cac tac acg cag aag agc 1394Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser445 450 455 460ctc tcc ctg tcc ccg ggt aaa
tgagtgaatt c 1426Leu Ser Leu Ser Pro Gly Lys 46517467PRTHomo
sapiens 17Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu
Gln Gly1 5 10 15Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr
Leu Gln Thr 20 25 30Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro
Ser Thr Leu Thr 35 40 45Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys
Asp Glu Ala Thr Ser 50 55 60Cys Ser Leu His Arg Ser Ala His Asn Ala
Thr His Ala Thr Tyr Thr65 70 75 80Cys His Met Asp Val Phe His Phe
Met Ala Asp Asp Ile Phe Ser Val 85 90 95Asn Ile Thr Asp Gln Ser Gly
Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105 110Leu Leu Ala Glu Ser
Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val 115 120 125Thr Phe Ser
Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp 130 135 140Pro
Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150
155 160Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu
Ile 165 170 175Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu
Phe Arg Lys 180 185 190Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly
Pro Met Pro Gly Ser 195 200 205Ser Tyr Gln Gly Thr Trp Ser Glu Trp
Ser Asp Pro Val Ile Phe Gln 210 215 220Thr Gln Ser Glu Glu Leu Lys
Glu Gly Trp Asn Gly Ser Gly Ser Arg225 230 235 240Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu Gly 245 250 255Ala Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265
270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 290 295 300His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu385 390
395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 405 410 415Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 420 425 430Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met 435 440 445His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 450 455 460Pro Gly Lys46518741DNAHomo
sapiensCDS(1)..(735) 18atg ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg
ctg ctg ctc cag gga 48Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu
Leu Leu Leu Gln Gly1 5 10 15ggc tgg ggc tgc ccc gac ctc gtc tgc tac
acc gat tac ctc cag acg 96Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr
Thr Asp Tyr Leu Gln Thr 20 25 30gtc atc tgc atc ctg gaa atg tgg aac
ctc cac ccc agc acg ctc acc 144Val Ile Cys Ile Leu Glu Met Trp Asn
Leu His Pro Ser Thr Leu Thr 35 40 45ctt acc tgg caa gac cag tat gaa
gag ctg aag gac gag gcc acc tcc 192Leu Thr Trp Gln Asp Gln Tyr Glu
Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60tgc agc ctc cac agg tcg gcc
cac aat gcc acg cat gcc acc tac acc 240Cys Ser Leu His Arg Ser Ala
His Asn Ala Thr His Ala Thr Tyr Thr65 70 75 80tgc cac atg gat gta
ttc cac ttc atg gcc gac gac att ttc agt gtc 288Cys His Met Asp Val
Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95aac atc aca gac
cag tct ggc aac tac tcc cag gag tgt ggc agc ttt 336Asn Ile Thr Asp
Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105 110ctc ctg
gct gag agc atc aag ccg gct ccc cct ttc aac gtg act gtg 384Leu Leu
Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val 115 120
125acc ttc tca gga cag tat aat atc tcc tgg cgc tca gat tac gaa gac
432Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp
130 135 140cct gcc ttc tac atg ctg aag ggc aag ctt cag tat gag ctg
cag tac 480Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu
Gln Tyr145 150 155 160agg aac cgg gga gac ccc tgg gct gtg agt ccg
agg aga aag ctg atc 528Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro
Arg Arg Lys Leu Ile 165 170 175tca gtg gac tca aga agt gtc tcc ctc
ctc ccc ctg gag ttc cgc aaa 576Ser Val Asp Ser Arg Ser Val Ser Leu
Leu Pro Leu Glu Phe Arg Lys 180 185 190gac tcg agc tat gag ctg cag
gtg cgg gca ggg ccc atg cct ggc tcc 624Asp Ser Ser Tyr Glu Leu Gln
Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205tcc tac cag ggg acc
tgg agt gaa tgg agt gac ccg gtc atc ttt cag 672Ser Tyr Gln Gly Thr
Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220acc cag tca
gag gag tta aag gaa ggc tgg aac aaa acc gaa acc tcc 720Thr Gln Ser
Glu Glu Leu Lys Glu Gly Trp Asn Lys Thr Glu Thr Ser225 230 235
240cag gtt gct ccg gca taatga 741Gln Val Ala Pro Ala
24519245PRTHomo sapiens 19Met Pro Arg Gly Trp Ala Ala Pro Leu Leu
Leu Leu Leu Leu Gln Gly1 5 10 15Gly Trp Gly Cys Pro Asp Leu Val Cys
Tyr Thr Asp Tyr Leu Gln Thr 20 25 30Val Ile Cys Ile Leu Glu Met Trp
Asn Leu His Pro Ser Thr Leu Thr 35 40 45Leu Thr Trp Gln Asp Gln Tyr
Glu Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60Cys Ser Leu His Arg Ser
Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75 80Cys His Met Asp
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95Asn Ile Thr
Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105 110Leu
Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val 115 120
125Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp
130 135 140Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu
Gln Tyr145 150 155 160Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro
Arg Arg Lys Leu Ile 165 170 175Ser Val Asp Ser Arg Ser Val Ser Leu
Leu Pro Leu Glu Phe Arg Lys 180 185 190Asp Ser Ser Tyr Glu Leu Gln
Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205Ser Tyr Gln Gly Thr
Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220Thr Gln Ser
Glu Glu Leu Lys Glu Gly Trp Asn Lys Thr Glu Thr Ser225 230 235
240Gln Val Ala Pro Ala 245201413DNAHomo sapiensCDS(1)..(1410) 20atg
ccg cgt ggc tgg gcc gcc ccc ttg ctc ctg ctg ctg ctc cag gga 48Met
Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5 10
15ggc tgg ggc tgc ccc gac ctc gtc tgc tac acc gat tac ctc cag acg
96Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr
20 25 30gtc atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc acg ctc
acc 144Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu
Thr 35 40 45ctt acc tgg caa gac cag tat gaa gag ctg aag gac gag gcc
acc tcc 192Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala
Thr Ser 50 55 60tgc agc ctc cac agg tcg gcc cac aat gcc acg cat gcc
acc tac acc 240Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala
Thr Tyr Thr65 70 75 80tgc cac atg gat gta ttc cac ttc atg gcc gac
gac att ttc agt gtc 288Cys His Met Asp Val Phe His Phe Met Ala Asp
Asp Ile Phe Ser Val 85 90 95aac atc aca gac cag tct ggc aac tac tcc
cag gag tgt ggc agc ttt 336Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser
Gln Glu Cys Gly Ser Phe 100 105 110ctc ctg gct gag agc atc aag ccg
gct ccc cct ttc aac gtg act gtg 384Leu Leu Ala Glu Ser Ile Lys Pro
Ala Pro Pro Phe Asn Val Thr Val 115 120 125acc ttc tca gga cag tat
aat atc tcc tgg cgc tca gat tac gaa gac 432Thr Phe Ser Gly Gln Tyr
Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp 130 135 140cct gcc ttc tac
atg ctg aag ggc aag ctt cag tat gag ctg cag tac 480Pro Ala Phe Tyr
Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160agg
aac cgg gga gac ccc tgg gct gtg agt ccg agg aga aag ctg atc 528Arg
Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile 165 170
175tca gtg gac tca aga agt gtc tcc ctc ctc ccc ctg gag ttc cgc aaa
576Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys
180 185 190gac tcg agc tat gag ctg cag gtg cgg gca ggg ccc atg cct
ggc tcc 624Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro
Gly Ser 195 200 205tcc tac cag ggg acc tgg agt gaa tgg agt gac ccg
gtc atc ttt cag 672Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro
Val Ile Phe Gln 210 215 220acc cag tca gag gag tta aag gaa ggc tgg
aac gat gac gat gac aag 720Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp
Asn Asp Asp Asp Asp Lys225 230 235 240ggc tcc ggc gac aaa act cac
aca tgc cca ccg tgc cca gca cct gaa 768Gly Ser Gly Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255gcc ctg ggg gca ccg
tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816Ala Leu Gly Ala Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270acc ctc atg
atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285gtg
agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295
300gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac
960Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn305 310 315 320agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg
cac cag gac tgg 1008Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp 325 330 335ctg aat ggc aag gag tac aag tgc aag gtc
tcc aac aaa gcc ctc cca 1056Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro 340 345 350gcc ccc atc gag aaa acc atc tcc
aaa gcc aaa ggg cag ccc cga gaa 1104Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365cca cag gtg tac acc ctg
ccc cca tcc cgg gag gag atg acc aag aac 1152Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380cag gtc agc ctg
acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile385 390 395 400gcc
gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1248Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410
415acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc aag
1296Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
420 425 430ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc
tca tgc 1344Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 435 440 445tcc gtg atg cat gag gct ctg cac aac cac tac acg
cag aag agc ctc 1392Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu 450 455 460tcc ctg tcc ccg ggt aaa tga 1413Ser Leu
Ser Pro Gly Lys465 47021470PRTHomo sapiens 21Met Pro Arg Gly Trp
Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly1 5 10 15Gly Trp Gly Cys
Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr 20 25 30Val Ile Cys
Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr 35 40 45Leu Thr
Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60Cys
Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr65 70 75
80Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val
85
90 95Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser
Phe 100 105 110Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn
Val Thr Val 115 120 125Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser Asp Tyr Glu Asp 130 135 140Pro Ala Phe Tyr Met Leu Lys Gly Lys
Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160Arg Asn Arg Gly Asp Pro
Trp Ala Val Ser Pro Arg Arg Lys Leu Ile 165 170 175Ser Val Asp Ser
Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190Asp Ser
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200
205Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln
210 215 220Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Asp Asp Asp
Asp Lys225 230 235 240Gly Ser Gly Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu 245 250 255Ala Leu Gly Ala Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 260 265 270Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 275 280 285Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn305 310 315
320Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
325 330 335Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro 340 345 350Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu 355 360 365Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn 370 375 380Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile385 390 395 400Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440
445Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
450 455 460Ser Leu Ser Pro Gly Lys465 470221754DNAMus
musculusCDS(1)..(720) 22atg ccc cgg ggc cca gtg gct gcc tta ctc ctg
ctg att ctc cat gga 48Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu
Leu Ile Leu His Gly1 5 10 15gct tgg agc tgc ctg gac ctc act tgc tac
act gac tac ctc tgg acc 96Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr
Thr Asp Tyr Leu Trp Thr 20 25 30atc acc tgt gtc ctg gag aca cgg agc
ccc aac ccc agc ata ctc agt 144Ile Thr Cys Val Leu Glu Thr Arg Ser
Pro Asn Pro Ser Ile Leu Ser 35 40 45ctc acc tgg caa gat gaa tat gag
gaa ctt cag gac caa gag acc ttc 192Leu Thr Trp Gln Asp Glu Tyr Glu
Glu Leu Gln Asp Gln Glu Thr Phe 50 55 60tgc agc cta cac agg tct ggc
cac aac acc aca cat ata tgg tac acg 240Cys Ser Leu His Arg Ser Gly
His Asn Thr Thr His Ile Trp Tyr Thr65 70 75 80tgc cat atg cgc ttg
tct caa ttc ctg tcc gat gaa gtt ttc att gtc 288Cys His Met Arg Leu
Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val 85 90 95aat gtg acg gac
cag tct ggc aac aac tcc caa gag tgt ggc agc ttt 336Asn Val Thr Asp
Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser Phe 100 105 110gtc ctg
gct gag agc atc aaa cca gct ccc ccc ttg aac gtg act gtg 384Val Leu
Ala Glu Ser Ile Lys Pro Ala Pro Pro Leu Asn Val Thr Val 115 120
125gcc ttc tca gga cgc tat gat atc tcc tgg gac tca gct tat gac gaa
432Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu
130 135 140ccc tcc aac tac gtg ctg agg ggc aag cta caa tat gag ctg
cag tat 480Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu
Gln Tyr145 150 155 160cgg aac ctc aga gac ccc tat gct gtg agg ccg
gtg acc aag ctg atc 528Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro
Val Thr Lys Leu Ile 165 170 175tca gtg gac tca aga aac gtc tct ctt
ctc cct gaa gag ttc cac aaa 576Ser Val Asp Ser Arg Asn Val Ser Leu
Leu Pro Glu Glu Phe His Lys 180 185 190gat tct agc tac cag ctg cag
gtg cgg gca gcg cct cag cca ggc act 624Asp Ser Ser Tyr Gln Leu Gln
Val Arg Ala Ala Pro Gln Pro Gly Thr 195 200 205tca ttc agg ggg acc
tgg agt gag tgg agt gac ccc gtc atc ttt cag 672Ser Phe Arg Gly Thr
Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220acc cag gct
ggg gag ccc gag gca ggc tgg gac ggc tcc ggc tct aga 720Thr Gln Ala
Gly Glu Pro Glu Ala Gly Trp Asp Gly Ser Gly Ser Arg225 230 235
240gagccccgcg gaccgacaat caagccctgt cctccatgca aatgcccagg
taagtcacta 780gaccagagct ccactcccgg gagaatggta agtgctataa
acatccctgc actagaggat 840aagccatgta cagatccatt tccatctctc
ctcatcagca cctaacctcg agggtggacc 900atccgtcttc atcttccctc
caaagatcaa ggatgtactc atgatctccc tgagccccat 960agtcacatgt
gtggtggtgg atgtgagcga ggatgaccca gatgtccaga tcagctggtt
1020tgtgaacaac gtggaagtac acacagctca gacacaaacc catagagagg
attacaacag 1080tactctccgg gtggtcagtg ccctccccat ccagcaccag
gactggatga gtggcaaggc 1140tttcgcatgc gccgtcaaca acaaagacct
cccagcgccc atcgagagaa ccatctcaaa 1200acccaaaggt gagagctgca
gcctgactgc atgggggctg ggatgggcat aaggataaag 1260gtctgtgtgg
acagccttct gcttcagcca tgacctttgt gtatgtttct accctcacag
1320ggtcagtaag agctccacag gtatatgtct tgcctccacc agaagaagag
atgactaaga 1380aacaggtcac tctgacctgc atggtcacag acttcatgcc
tgaagacatt tacgtggagt 1440ggaccaacaa cgggaaaaca gagctaaact
acaagaacac tgaaccagtc ctggactctg 1500atggttctta cttcatgtac
agcaagctga gagtggaaaa gaagaactgg gtggaaagaa 1560atagctactc
ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg actaagagct
1620tctcccggac tccgggtaaa tgagctcagc acccacaaaa ctctcaggtc
caaagagaca 1680cccacactca tctccatgct tcccttgtat aaataaagca
cccagcaatg cctgggacca 1740tgtaatagga attc 175423240PRTMus musculus
23Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly1
5 10 15Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp
Thr 20 25 30Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile
Leu Ser 35 40 45Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln
Glu Thr Phe 50 55 60Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His
Ile Trp Tyr Thr65 70 75 80Cys His Met Arg Leu Ser Gln Phe Leu Ser
Asp Glu Val Phe Ile Val 85 90 95Asn Val Thr Asp Gln Ser Gly Asn Asn
Ser Gln Glu Cys Gly Ser Phe 100 105 110Val Leu Ala Glu Ser Ile Lys
Pro Ala Pro Pro Leu Asn Val Thr Val 115 120 125Ala Phe Ser Gly Arg
Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu 130 135 140Pro Ser Asn
Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155
160Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile
165 170 175Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe
His Lys 180 185 190Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro
Gln Pro Gly Thr 195 200 205Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser
Asp Pro Val Ile Phe Gln 210 215 220Thr Gln Ala Gly Glu Pro Glu Ala
Gly Trp Asp Gly Ser Gly Ser Arg225 230 235 24024795DNAMus
musculusCDS(19)..(783) 24ctgcaggtcg acaccacc atg ccc cgg ggc cca
gtg gct gcc tta ctc ctg 51 Met Pro Arg Gly Pro Val Ala Ala Leu Leu
Leu 1 5 10ctg att ctc cat gga gct tgg agc tgc ctg gac ctc act tgc
tac act 99Leu Ile Leu His Gly Ala Trp Ser Cys Leu Asp Leu Thr Cys
Tyr Thr 15 20 25gac tac ctc tgg acc atc acc tgt gtc ctg gag aca cgg
agc ccc aac 147Asp Tyr Leu Trp Thr Ile Thr Cys Val Leu Glu Thr Arg
Ser Pro Asn 30 35 40ccc agc ata ctc agt ctc acc tgg caa gat gaa tat
gag gaa ctt cag 195Pro Ser Ile Leu Ser Leu Thr Trp Gln Asp Glu Tyr
Glu Glu Leu Gln 45 50 55gac caa gag acc ttc tgc agc cta cac agg tct
ggc cac aac acc aca 243Asp Gln Glu Thr Phe Cys Ser Leu His Arg Ser
Gly His Asn Thr Thr60 65 70 75cat ata tgg tac acg tgc cat atg cgc
ttg tct caa ttc ctg tcc gat 291His Ile Trp Tyr Thr Cys His Met Arg
Leu Ser Gln Phe Leu Ser Asp 80 85 90gaa gtt ttc att gtc aat gtg acg
gac cag tct ggc aac aac tcc caa 339Glu Val Phe Ile Val Asn Val Thr
Asp Gln Ser Gly Asn Asn Ser Gln 95 100 105gag tgt ggc agc ttt gtc
ctg gct gag agc atc aaa cca gct ccc ccc 387Glu Cys Gly Ser Phe Val
Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro 110 115 120ttg aac gtg act
gtg gcc ttc tca gga cgc tat gat atc tcc tgg gac 435Leu Asn Val Thr
Val Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp 125 130 135tca gct
tat gac gaa ccc tcc aac tac gtg ctg agg ggc aag cta caa 483Ser Ala
Tyr Asp Glu Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln140 145 150
155tat gag ctg cag tat cgg aac ctc aga gac ccc tat gct gtg agg ccg
531Tyr Glu Leu Gln Tyr Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro
160 165 170gtg acc aag ctg atc tca gtg gac tca aga aac gtc tct ctt
ctc cct 579Val Thr Lys Leu Ile Ser Val Asp Ser Arg Asn Val Ser Leu
Leu Pro 175 180 185gaa gag ttc cac aaa gat tct agc tac cag ctg cag
gtg cgg gca gcg 627Glu Glu Phe His Lys Asp Ser Ser Tyr Gln Leu Gln
Val Arg Ala Ala 190 195 200cct cag cca ggc act tca ttc agg ggg acc
tgg agt gag tgg agt gac 675Pro Gln Pro Gly Thr Ser Phe Arg Gly Thr
Trp Ser Glu Trp Ser Asp 205 210 215ccc gtc atc ttt cag acc cag gct
ggg gag ccc gag gca ggc tgg gac 723Pro Val Ile Phe Gln Thr Gln Ala
Gly Glu Pro Glu Ala Gly Trp Asp220 225 230 235ggc agc gga cac cac
cat cat cac cac ggt agc ggc gac tat aaa gac 771Gly Ser Gly His His
His His His His Gly Ser Gly Asp Tyr Lys Asp 240 245 250gat gac gat
aag tagtgagaat tc 795Asp Asp Asp Lys 25525255PRTMus musculus 25Met
Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly1 5 10
15Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr
20 25 30Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu
Ser 35 40 45Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu
Thr Phe 50 55 60Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His Ile
Trp Tyr Thr65 70 75 80Cys His Met Arg Leu Ser Gln Phe Leu Ser Asp
Glu Val Phe Ile Val 85 90 95Asn Val Thr Asp Gln Ser Gly Asn Asn Ser
Gln Glu Cys Gly Ser Phe 100 105 110Val Leu Ala Glu Ser Ile Lys Pro
Ala Pro Pro Leu Asn Val Thr Val 115 120 125Ala Phe Ser Gly Arg Tyr
Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu 130 135 140Pro Ser Asn Tyr
Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr145 150 155 160Arg
Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile 165 170
175Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys
180 185 190Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro Gln Pro
Gly Thr 195 200 205Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro
Val Ile Phe Gln 210 215 220Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp
Asp Gly Ser Gly His His225 230 235 240His His His His Gly Ser Gly
Asp Tyr Lys Asp Asp Asp Asp Lys 245 250 25526792DNAMus
musculusCDS(1)..(786) 26atg aaa ttc tta gtc aac gtt gcc ctt gtt ttt
atg gtc gtg tac att 48Met Lys Phe Leu Val Asn Val Ala Leu Val Phe
Met Val Val Tyr Ile1 5 10 15tct tac atc tat gcc ggc agc gga cac cac
cat cat cac cac ggt agc 96Ser Tyr Ile Tyr Ala Gly Ser Gly His His
His His His His Gly Ser 20 25 30ggc gac tat aaa gac gat gac gat aag
ggt tcc gga tgc ctg gac ctc 144Gly Asp Tyr Lys Asp Asp Asp Asp Lys
Gly Ser Gly Cys Leu Asp Leu 35 40 45act tgc tac act gac tac ctc tgg
acc atc acc tgt gtc ctg gag aca 192Thr Cys Tyr Thr Asp Tyr Leu Trp
Thr Ile Thr Cys Val Leu Glu Thr 50 55 60cgg agc ccc aac ccc agc ata
ctc agt ctc acc tgg caa gat gaa tat 240Arg Ser Pro Asn Pro Ser Ile
Leu Ser Leu Thr Trp Gln Asp Glu Tyr65 70 75 80gag gaa ctt cag gac
caa gag acc ttc tgc agc cta cac agg tct ggc 288Glu Glu Leu Gln Asp
Gln Glu Thr Phe Cys Ser Leu His Arg Ser Gly 85 90 95cac aac acc aca
cat ata tgg tac acg tgc cat atg cgc ttg tct caa 336His Asn Thr Thr
His Ile Trp Tyr Thr Cys His Met Arg Leu Ser Gln 100 105 110ttc ctg
tcc gat gaa gtt ttc att gtc aat gtg acg gac cag tct ggc 384Phe Leu
Ser Asp Glu Val Phe Ile Val Asn Val Thr Asp Gln Ser Gly 115 120
125aac aac tcc caa gag tgt ggc agc ttt gtc ctg gct gag agc atc aaa
432Asn Asn Ser Gln Glu Cys Gly Ser Phe Val Leu Ala Glu Ser Ile Lys
130 135 140cca gct ccc ccc ttg aac gtg act gtg gcc ttc tca gga cgc
tat gat 480Pro Ala Pro Pro Leu Asn Val Thr Val Ala Phe Ser Gly Arg
Tyr Asp145 150 155 160atc tcc tgg gac tca gct tat gac gaa ccc tcc
aac tac gtg ctg agg 528Ile Ser Trp Asp Ser Ala Tyr Asp Glu Pro Ser
Asn Tyr Val Leu Arg 165 170 175ggc aag cta caa tat gag ctg cag tat
cgg aac ctc aga gac ccc tat 576Gly Lys Leu Gln Tyr Glu Leu Gln Tyr
Arg Asn Leu Arg Asp Pro Tyr 180 185 190gct gtg agg ccg gtg acc aag
ctg atc tca gtg gac tca aga aac gtc 624Ala Val Arg Pro Val Thr Lys
Leu Ile Ser Val Asp Ser Arg Asn Val 195 200 205tct ctt ctc cct gaa
gag ttc cac aaa gat tct agc tac cag ctg cag 672Ser Leu Leu Pro Glu
Glu Phe His Lys Asp Ser Ser Tyr Gln Leu Gln 210 215 220gtg cgg gca
gcg cct cag cca ggc act tca ttc agg ggg acc tgg agt 720Val Arg Ala
Ala Pro Gln Pro Gly Thr Ser Phe Arg Gly Thr Trp Ser225 230 235
240gag tgg agt gac ccc gtc atc ttt cag acc cag gct ggg gag ccc gag
768Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ala Gly Glu Pro Glu
245 250 255gca ggc tgg gac tag tga gaattc 792Ala Gly Trp Asp
26027260PRTMus musculus 27Met Lys Phe Leu Val Asn Val Ala Leu Val
Phe Met Val Val Tyr Ile1 5 10 15Ser Tyr Ile Tyr Ala Gly Ser Gly His
His His His His His Gly Ser 20 25 30Gly Asp Tyr Lys Asp Asp Asp Asp
Lys Gly Ser Gly Cys Leu Asp Leu 35 40 45Thr Cys Tyr Thr Asp Tyr Leu
Trp Thr Ile Thr Cys Val Leu Glu Thr 50 55 60Arg Ser Pro Asn Pro Ser
Ile Leu Ser Leu Thr Trp Gln Asp Glu Tyr65 70 75 80Glu Glu Leu Gln
Asp Gln Glu Thr Phe Cys Ser Leu His Arg Ser Gly 85 90 95His Asn Thr
Thr His Ile Trp Tyr Thr Cys His Met Arg Leu Ser Gln 100 105 110Phe
Leu Ser Asp Glu Val Phe Ile Val Asn Val Thr Asp Gln Ser Gly 115 120
125Asn Asn Ser Gln Glu Cys Gly Ser Phe Val Leu Ala Glu Ser Ile Lys
130 135 140Pro Ala Pro Pro Leu Asn Val Thr Val Ala Phe Ser Gly Arg
Tyr Asp145 150 155 160Ile Ser Trp Asp Ser Ala Tyr
Asp Glu Pro Ser Asn Tyr Val Leu Arg 165 170 175Gly Lys Leu Gln Tyr
Glu Leu Gln Tyr Arg Asn Leu Arg Asp Pro Tyr 180 185 190Ala Val Arg
Pro Val Thr Lys Leu Ile Ser Val Asp Ser Arg Asn Val 195 200 205Ser
Leu Leu Pro Glu Glu Phe His Lys Asp Ser Ser Tyr Gln Leu Gln 210 215
220Val Arg Ala Ala Pro Gln Pro Gly Thr Ser Phe Arg Gly Thr Trp
Ser225 230 235 240Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ala
Gly Glu Pro Glu 245 250 255Ala Gly Trp Asp 2602820DNAArtificialPCR
Primer 28gccagatcgc ctcctgatta 202920DNAArtificialPCR Primer
29catgctcaca gtgccccttt 203020DNAArtificialPCR Primer 30ctcccccctt
gaacgtgact 203120DNAArtificialPCR Primer 31ttgcccctca gcacgtagtt
203228DNAArtificialPCR Primer 32agagccagat tatctctttc tacctcag
283320DNAArtificialPCR Primer 33cctttttcgc cttgctgttg
203440PRTartificialpeptide linker 34Ser Gly Gly Gly Gly Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 35 4035551PRTHomo sapiens 35Met Ala Ala Pro Ala Leu Ser Trp
Arg Leu Pro Leu Leu Ile Leu Leu1 5 10 15Leu Pro Leu Ala Thr Ser Trp
Ala Ser Ala Ala Val Asn Gly Thr Ser 20 25 30Gln Phe Thr Cys Phe Tyr
Asn Ser Arg Ala Asn Ile Ser Cys Val Trp 35 40 45Ser Gln Asp Gly Ala
Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp 50 55 60Pro Asp Arg Arg
Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser65 70 75 80Gln Ala
Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln 85 90 95Lys
Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu 100 105
110Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu
115 120 125Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His
Val Glu 130 135 140Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln
Ala Ser His Tyr145 150 155 160Phe Glu Arg His Leu Glu Phe Glu Ala
Arg Thr Leu Ser Pro Gly His 165 170 175Thr Trp Glu Glu Ala Pro Leu
Leu Thr Leu Lys Gln Lys Gln Glu Trp 180 185 190Ile Cys Leu Glu Thr
Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val 195 200 205Arg Val Lys
Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser 210 215 220Gln
Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr225 230
235 240Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe
Gly 245 250 255Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn
Thr Gly Pro 260 265 270Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro
Asp Pro Ser Lys Phe 275 280 285Phe Ser Gln Leu Ser Ser Glu His Gly
Gly Asp Val Gln Lys Trp Leu 290 295 300Ser Ser Pro Phe Pro Ser Ser
Ser Phe Ser Pro Gly Gly Leu Ala Pro305 310 315 320Glu Ile Ser Pro
Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu 325 330 335Leu Leu
Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn 340 345
350His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His
355 360 365Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe
Thr Tyr 370 375 380Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val
Ala Gly Ala Pro385 390 395 400Thr Gly Ser Ser Pro Gln Pro Leu Gln
Pro Leu Ser Gly Glu Asp Asp 405 410 415Ala Tyr Cys Thr Phe Pro Ser
Arg Asp Asp Leu Leu Leu Phe Ser Pro 420 425 430Ser Leu Leu Gly Gly
Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser 435 440 445Gly Ala Gly
Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro 450 455 460Arg
Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro465 470
475 480Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu
Ala 485 490 495Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val
Ser Phe Pro 500 505 510Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg
Ala Leu Asn Ala Arg 515 520 525Leu Pro Leu Asn Thr Asp Ala Tyr Leu
Ser Leu Gln Glu Leu Gln Gly 530 535 540Gln Asp Pro Thr His Leu
Val545 550
* * * * *
References